bluuebunny/crossref_metadata_2025 huggingface dataset:
cAMP and cGMP in nasal mucus related to severity of smell loss in patients with smell dysfunction
Authors: Henkin R I , Velicu I
Published: May 2017

Purpose: To evaluate nasal mucus levels of cAMP and cGMP in patients with taste and smell dysfunction with respect to severity of their smell loss.

Methods: cAMP and cGMP were measured in nasal mucus using a sensitive spectrophotometric 96 plate ELISA technique. Smell loss was measured in patients with taste and smell dysfunction by standardized psychophysical measurements of olfactory function and classified by severity of loss into four types from most severe to least severe such that anosmia > Type I hyposmia > Type II hyposmia > Type III hyposmia. Measurements of nasal mucus cyclic nucleotides and smell loss were made independently.

Results: As smell loss severity increased stepwise cAMP and cGMP levels decreased stepwise [cAMP, cGMP (in pmol/ml); anosmia – 0.004, 0.008: Type I hyposmia – 0.12±0.03, 0.10±0.03: Type II hyposmia – 0.15±0.02, 0.16±0.01: Type III hyposmia – 0.23±0.05, 0.20±0.15].

Conclusions: These results confirm the association of biochemical changes in cyclic nucleotides with systematic losses of smell acuity. These results confirm the usefulness of the psychophysical methods we defined to determine the systematic classification of smell loss severity. These changes can form the basis for the biochemical definition of smell loss among some patients with smell loss as well as for their therapy.
Change and significance of the expression of c-kit and SCF following recovery from unilateral testicular torsion in rats
Authors: Guan Yi , Zheng XinMin , Yang ZhiWei , Li ShiWen
Published: May 2017

Purpose: To investigate the change in expression levels of c-kit and SCF, and the protective effects of FSH on ischemia-reperfusion injury due to testicular torsion-detorsion.

Methods: 24 adult male SD rats were divided into three groups of 8: control group, testicular torsion group and FSH-treated group. The control group was treated with sham-operation. Animals in the testicular torsion and FSH-treated groups were subjected to unilateral 720°counterclockwise testicular torsion for 2 hours and then reperfusion was allowed after detorsion. The FSH-treated group received intraperitoneal injection of FSH 15min before detorsion. Then, the rats were sacrificed and the testes were harvested. Histopathological changes were observed by light microscope, and the expression levels of c-kit, SCF in testicular tissue in the different groups were detected by Immunohistochemical assay and Quantitative Real-time RT-PCR analysis. Finally, the relative proportions of germ cells were measured by FCM.

Results: c-kit and SCF were positive expressed in 52.58% and 61.16% of testicular cells of control tissues, respectively. Decreases of c-kit and SCF positive cells (15.01% and 9.18%) were found in the testicular torsion group. After being treated by FSH, the number of positive cells increased (31.25% and 20.01%). Moreover, the c-kit and SCF mRNA expression was increased dramatically (P < 0.01) in response to FSH stimulation. Furthermore, the number of haploid, diploid and tetraploid cells has also increased significantly in drug-treated testes (P < 0.01).

Conclusion: The mechanism of tissue damage in the testicular torsion model, includes changes in the expression of c-kit and SCF following torsion. Also, FSH has a protective effect on germ cells after unilateral testicular torsion, which was reflected by increased c-kit and SCF levels.
Hyaluronan oligosaccharides are potential stimulators to angiogenesis via RHAMM mediated signal pathway in wound healing
Authors: Gao F. , Yang C. X. , Mo W. , Liu Y. W. , He Y. Q.
Published: May 2017

Purpose: To determine if oligosaccharides of hyaluronan (o-HA) promotes wound recovery by accelerating angiogenesis and to study the mechanisms by which o-HA stimulates endothelial cell (EC) proliferation.

Methods: Using hyaluronidase digestion, we prepared a mixture of hyaluronan (HA) fragments sizesd 2 to 10 disaccharides units, and studied their effects on EC growth and migration in mimicking wound recovery in vitro. The effects of o-HA on EC growth in vitro were studied by counting cell numbers. The roles of 2 hyaluronan receptors on EC cells, CD44 and RHAMM (Receptor for HA-Mediated Motility), were studied in initiating signaling cascades, using immunoblot assay. Signal transduction was determined by blocking antibodies to CD44 and RHAMM. An in vitro wound healing model was prepared by scratching the cellular layer of cultured EC, and movement of cells into the denuded area was quantified.

Results: o-HA was a strong stimulator to EC proliferation at low concentration 10µg/ml compared with native high molecular weight HA (n-HA) (P < 0.01). Signal transduction may be initiated by o-HA via RHAMM receptor on EC membrane, but not CD44. In the in vitro model, the lesion area was nearly completely recovered when the EC layer was exposed to o-HA 40hrs post-injury, whereas the wound area remained half recovered pretreated with native undegraded large HA and control medium.(P < 0.05 from 24 to 40hrs).

Conclusion: Hyaluronan oligosaccharides may play a role in wound healing by increasing angiogenesis. o-HA-RHAMM binding dependent signal transduction pathway may be important in the regulation of angiogenesis associated with EC proliferation.
The relationship between cervical human papillomavirus infection and apoptosis
Authors: Fidan Isil , Bozdayi Gulendam , Rota Seyyal , Biri Aydan , Gurelik Feryal Çetin , Yuksel Sevgi , Imir Turgut
Published: May 2017

Purpose: Cervical carcinoma is the second most common cancer among women worldwide. Viral infections, especially human papillomavirus (HPV) infections, are important factors in its etiology. Changes in apoptotic regulation are considered to have an important role in the carcinogenesis development. In this study, the relationship between apoptosis and HPV infection was investigated.

Methods: HPV DNA and HPV DNA type 16 positivity were detected in 110 cervical smear samples with Real Time PCR and sequencing was performed for HPV DNA type 18. The presence of apoptosis was investigated using TUNEL and Annexin V staining methods and analyzed by fluorescence microscope and flow cytometry.

Results: HPV DNA type 16 was detected in 9 samples (8.1%), HPV DNA type 18 positive in 6 samples (5.4%) and HPV types other than HPV type 16 and HPV type 18 in 9 samples (8.1%). A decrease apoptosis was found in HPV DNA positive samples compared with controls (P < 0.05).

Conclusion: The decrease of apoptosis during HPV infection might cause cellular immortality and then malignant transformation.
Phaeochromocytoma comb?ned with subclinical Cushing's syndrome and pituitary microadenoma
Authors: Yaylali Guzin Fidan , Akin Fulya , Bastemir Mehmet , Yaylali Yalin Tolga , Ozden Akin
Published: May 2017

Objectives: Phaeochromocytoma (PHEO) occasionally associates with pathological lesions of the adrenal cortex. The coexistence of PHEO and pre-clinical Cushing's syndrome (PCS) of the same adrenal gland has rarely been reported. We report a case of PHEO and PCS originating from the same adrenal gland and discuss the peculiar diagnostic aspects of this entity.

Clinical Presentation: A 64 yr old man was hospitalized to evaluate the right adrenal mass which was discovered incidentally by ultrasonography. He had a history of type 2 diabetes mellitus and hyperlipidemia. Blood pressure measurements were all normal during his hospital stay. Laboratory examination showed: urinary catecholamines were markedly increased. HbA1C of 14.3 %, midnight cortisol of 11(?g/dL), cortisol was not suppressed after the overnight 1 mg oral dexamethasone suppression test (DST): 3.42(?g/dL), 24 hr free cortisol in the urine : 213 µg/day (10-100), cortisol levels were suppressed more than 50% with 8 mg of dexamethasone. CT scan of the adrenal glands showed a 6 cm well encapsulated right adrenal mass together with a clearly normal left adrenal gland. MRI investigation of the sella turcica revealed a pituitary microadenoma on the right side of the adenohypophysis He was treated with ? and subsequent ? blockers after the diagnosis of PHEO and PCS was made. Right adrenalectomy was performed. The pathology showed typical PHEO with adrenocortical hyperplasia. VMA, metanefrin and free cortisol levels were normalized one month after surgery.

Conclusion: The present report is a rare case of PHEO combined with PCS in the same adrenal gland.
Aminoguanidine ameliorates radiation-induced oxidative lung damage in rats
Authors: Eroglu Celalettin , Yildiz Oguz Galip , Saraymen Recep , Soyuer Serdar , Kilic Eser , Ozcan Servet
Published: May 2017

Purpose: To investigate the possible protective effects of aminoguanidine (AG ) on lung damage in whole body irradiated rats.

Methods: To evaluate the biological damage of radiation on rat lung tissue, lipid peroxidation products were measured using biochemical parameters. Thirty Wistar albino rats were divided into three subgroups: control (C) , irradiation alone (RT), and RT + AG combined. After sacrificing the rats, antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities and malondiadehyde (MDA), nitric oxide (NO) levels were evaluated in lung tissue.

Results: Administration of AG resulted in an increase in the activities of CAT, SOD and GSHPx in the lungs. All were reduced after radiatio. In addition, AG administration resulted in a decrease in both NO and MDA levels in lung compared with the irradiated group.

Conclusion: Amnoguanidine increased the endogenous antioxidant defence mechanism in rats and protected the animals from radiation-induced lung toxicity. Moreover, AG may protect against ionizing radiation-induced lung damage because of its antioxidant effect.
Clinicopathological evaluation of immunohistochemical Ki-67 and endothelial nitric oxide synthase expression in intracranial ependymoma
Authors: Yan Xuetao , Cheng Xiaoli , Liu Juyin , Luo Dongqin , He Xianghu , Chen Feng , Qin Chengming , Wang Yanlin
Published: May 2017

Purpose: To analyze the association between Ki-67 and eNOS expression with the pathological grades of patients with intracranial ependymomas, and to determine its value in distinguishing the progression of the disease.

Methods: A clinicopathological study was undertaken in 82 patients with intracranial ependymomas. Tissue samples, obtained by tumour resection, were divided into three groups: low-grade, mid-grade and high-grade ependymomas. Tissue samples obtained from 15 patients with brain contusion were used as control. Immuno-histochemical staining was performed to analyze the association between Ki-67 and eNOS expression with various tumour grades. The cell proliferating marker Ki-67 was assessed by positive cell count. The levels of eNOS positive expression were evaluated as slight, moderate and intense.

Results: 48 of 82 cases (58.54%) expressed Ki-67 protein. Expression of Ki-67 and eNOS was negative in all control samples. Positive cell rates were 2.65±0.83 % in the low-grade, 9.63±0.08 % in the mid-grade, and 28.41±0.71 % in the high-grade ependymoma groups. In low-grade ependymomas there were 8 and 12 cases that expressed eNOS slightly or moderately. In the mid-grade ependymoma group eNOS was expressed moderately in 10 cases and intensely in 15. In the high-grade group 20 cases showed intense positive expression of eNOS. The Ki-67 positive cell counts for slight, moderate and intense eNOS expression were 2.20, 6.07 and 22.25, respectively.

Conclusion: Ki-67 and eNOS expression in intracranial ependymoma tissue was associated with the histopathological grade and malignant degree.
Protective effect of ganoderan on renal damage in rats with chronic glomerulonephritis
Authors: Zhong Wei-De , He Hui-Chan , Ou Ru-Biao , Bi Xue-Cheng , Dai Qi-Shan , Han Zhao-Dong , Liang Yu-Xiang , Ye Yong-Kan , Qin Wei-Jun , Li Ze , Zeng Guo-hua , Zhu Gang
Published: May 2017

Purpose: To investigate the protective effect of ganoderan on renal damage in rat models with chronic glomerulonephritis induced by adriamycin.

Methods: 48 healthy Sprague-Dawley rats were randomly divided into three groups: control, nephritic model and ganoderan treatment groups. Changes of the following indices in the three groups were observed 6 weeks after treatment: 24-hour urine protein, albumen, serum creatinine, cholesterol. Histopathological observations of the renal cortex were made by light and electron microscopy.

Results: Compared with controls, levels of 24-hour urine protein (9.60±0.57mg/d vs. 82.50±3.18mg/d), serum creatinine (35.25±2.63?mol/L vs. 44.75±8.06?mol/L) and cholesterol (1.15±0.10mmol/L vs. 4.02±0.25mmol/L) of rats in the nephritic model group were increased (P < 0.05), and the concentration of albumen was decreased (35.98±1.34g/L vs. 19.05±0.62g/L, P < 0.05). Ganoderan administration decreased 24-hour urine protein (82.50±3.18mg/d vs. 45.01±3.94mg/d, P < 0.05). Following ganoderan, the pathological changes in kidney tissue were improved compared with those in the nephritic model group.

Conclusion: Ganoderan exerts protective effects in rats with chronic glomerulonephritis induced by ADR. Ganoderan reduced 24-hour urine protein, serum creatinine, cholesterol, improving renal function and reducing the severity of renal injury.
OPTIMIZING VITAMIN D LEVELS IN PATIENTS WITH MULTIPLE SCLEROSIS
Authors: Ahluwalia P S , Metz L M , Hanley D A
Published: May 2017

Background: Emerging research suggests that vitamin D plays animportant role beyond bone health, particularly in immune function and may beimportant in people with Multiple Sclerosis (MS). MS clinic physicians in Calgary, Alberta believe that patients should have at least minimallysufficient ( > 80nmol/L) serum 25(OH)D levels to maintain adequate bone health. Involving patients in the assessment and management of their ownvitamin D needs may be effective and more efficient than having clinicians track levels.

Objectives: Determine the prevalence of vitamin D insufficiency and the feasibility of using different management methods to optimize serum25(OH)D levels.

Methods: 213 patients who attended the Calgary MS Clinicbetween September 2006 and January 2007 participated in this study. Eachpatient agreed to have serum 25(OH)D levels measured, and to adjust theirvitamin D dose according to an algorithm that they would follow or that would be used by a graduate student to recommend dose changes at baseline, 3- and 6-months.

Results: Mean age was 45.6 years (range 21-72); 78.9% werewomen. Mean EDSS was 3.2 (range 0-8.5). Mean baseline serum level was 72.8nmol/L (SD 26.8) (range 17.9-160.0); 62.4% had levels < 80 nmol/L. 60.6% of subjects were taking at least 1000 IU.

Conclusions: We found a high prevalence of vitamin D insufficiency despite a sizeable proportion taking at least 1000 IU of vitamin D3 daily. Six month data, including adherence to the study protocol and proportion ofparticipants with optimized 25(OH)D levels will be presented. Insight into themanagement of dosing for patients could have an impact on the integration ofvitamin D optimization into the MS population.
MOLECULAR CONTROL OF ARTICULAR CARTILAGE DEGENERATION BY TRANSFORMING GROWTH FACTOR ALPHA
Authors: Appleton Tom , Usmani Shirine , Mort John , Beier Frank
Published: May 2017

Background: Articular cartilage degeneration is a hallmark of osteoarthritis (OA). We previously identified increased expression of transforming growth factor alpha (TGF?) and chemokine (C-C motif) ligand 2 (CCL2) in articular cartilage from a rat modelof OA (1,2). We subsequently reported that TGF? signalling modified chondrocyte cytoskeletal organization, increased catabolic and decreased anabolic gene expression and suppressed Sox9. Due to other roles in chondrocytes, we hypothesized that the effects ofTGF? on chondrocytes are mediated by Rho/ROCK and MEK/ERK signaling pathways.

Methods: Primary cultures of chondrocytes and articularosteochondral explants were treated with pharmacological inhibitors of MEK1/2(U0126), ROCK (Y27632), Rho (C3), p38 MAPK (SB202190) and PI3K (LY294002) to elucidate pathway involvement.

Results: Using G-LISA we determined that stimulation of primary chondrocytes with TGF? activates RhoA. Reciprocally, inhibition of RhoA/ROCK but not other signalling pathways prevents modification of the actin cytoskeleton in responseto TGF?. Inhibition of MEK/ERKsignaling rescued suppression of anabolic gene expression by TGF? including SOX9 mRNA and protein levels. Inhibition of MEK/ERK, Rho/ROCK, p38 MAPK and PI3K signalling pathways differentially controlled the induction of MMP13 and TNF? gene expression. TGF? also induced expression of CCL2 specifically through MEK/ERK activation. In turn, CCL2 treatment induced the expression of MMP3 and TNF?. Finally, we assessed cartilage degradation by immunohistochemical detection of type II collagen cleavage fragments generated by MMPs. Blockade of RhoA/ROCK and MEK/ERK signalling pathways reduced the generation of type IIcollagen cleavage fragments in response to TGF? stimulation.

Conclusions: Rho/ROCK signalling mediates TGF?-induced changes inchondrocyte morphology, while MEK/ERK signalling mediates the suppression ofSox9 and its target genes, and CCL2 expression. CCL2, in turn, induces the expression of MMP3 and TNF?, two potent catabolic factors known to be involved in OA. These pathways may represent strategic targets for interventional approaches to treating cartilage degeneration in osteoarthritis.

References:

1. Appleton CTG et al. Arthritis Rheum 2007;56:1854-68.

2. Appleton CTG et al. Arthritis Rheum 2007; 56:3693-705.
THE TRANSCRIPTION FACTOR Y-BOX BINDING PROTEIN-1 (YB-1) INDUCES EXPRESSION OF THE PIK3CA ONCOGENE LEADING TO INCREASED INVASION OFBASAL-LIKE BREAST CARCINOMA CELLS
Authors: Astanehe Arezoo , Finkbeiner Melanie , To Karen , Dunn Sandra E
Published: May 2017

Background: Basal-like breast carcinoma (BLBC) is the mostaggressive subtype of breast cancer. 73% of BLBC over-express YB-1, anoncogenic transcription/translation factor. PIK3CA, which codes for the p110? catalytic subunit ofphosphatidylinositol-3-kinase (PI3K), is another oncogene. The PI3K signalling pathway is fundamental in the regulation of many cellular functions and isoften deregulated in cancer. Despite its importance, the knowledge on the transcriptional regulation of PIK3CA is limited. Indeed, we have recently published the first report on the PIK3CA promoter.

Methods and Results: A genome-wide chromatin immunoprecipitation on chip (ChIP-on-chip) analysis of a BLBC cell-line(SUM149) suggested binding of YB-1 to the PIK3CA promoter. This binding was verified using traditional chromatin immunoprecipitation (ChIP). Furthermore, electrophoretic mobility shift assay (EMSA) using oligonucleotides with eitherwild-type or mutated YB-1 responsive elements mapped YB-1 binding to three sites on the PIK3CA promoter. Silencing YB-1 in BLBC cell-lines (SUM149, HCC1937, andMDA-MB-231) decreased, while over-expression of YB-1 increased the PIK3CA promoter activity, transcript, and protein levels. Interestingly, array comparative genomic hybridization(aCGH) and quantitative PCR demonstrated PIK3CA copy number gains in HCC1937 andMDA-MB-231 cells. Although PIK3CA amplifications are overall uncommon (9%) in breast cancer, we demonstrated here that low level gains in PIK3CA copy number are present in 30%of primary BLBC cases. Furthermore, it has previously been demonstrated that mutations of PIK3CA are the most common genetic aberration (27%) found in breast cancer. These mutations lead to constitutive activation of p110? and are highly oncogenic. Over-expression of YB-1in MCF-7 cells, which harbour an activating PIK3CA mutation, increased PIK3CA transcript and protein levels. Furthermore, induction of PIK3CA by YB-1 leads to increased levels of urokinase plasminogenactivator (uPA) and invasion.

Conclusions: Our data demonstrates that YB-1 binds to the PIK3CA promoter and induces itsexpression whether the gene is wild-type or amplified. Moreover, since YB-1induces expression of the active mutant p110?, then therapeutic inhibition of YB-1 may lead to decreased p110? and interference with the constitutively activated PI3K pathway in cancers. In addition, the YB-1/PIK3CA/uPA network provides information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.

A.A. is supported by a Child and Family–CIHR–UBC MD/PhD Studentship Award.
THE IMPACT OF ESTIMATED GLOMERULAR FILTRATION RATEREPORTING ON NEPHROLOGY REFERRAL PATTERN, PATIENT CHARACTERISTICS AND OUTCOME
Authors: Bergman Arik , Naimark David MJ
Published: May 2017

Background: Chronic kidney disease (CKD) is a growing public health problem worldwide. The key to reducing the burden of CKD is earlydetection and delay of disease progression. An elevated serum creatinine concentration, [Cr], is acommon indicator of CKD. However, even with advanced CKD, [Cr] may not be highamong patients with low muscle mass (particularly the elderly). Thus, the use of the estimated glomerular filtration rate (eGFR) has been advocated for identifying severe occult kidney disease. In January 2006, community laboratories in Ontario, Canada, began to report an eGFR value along with every [Cr] result. The present study sought to investigate the impact of eGFR reporting on nephrology referrals and subsequent patient outcome.

Methods: The current study consisted of a retrospective analysis of all referrals to the adult general nephrology clinics at Sunnybrook Health Sciences Centre 15 months before and after eGFR reporting took effect on January 1, 2006.

Results: eGFR reporting was associated with a significant rise in the number of referrals (971 to 1101, p=0.03), a 51% rise in patient wait-time (from 73 days to 110days, p < 0.001) and an increase in nephrologists’ workload. Patients referred after eGFR reporting were older, but suffered from fewer comorbidities such as hypertension, vascular disease, and dementia. There was an increase in the number of patients referred with stage 3 CKD, but no change in the proportion of stage 4 and 5 CKD referrals or the number of patients initiating dialysis.

Conclusion: Laboratory reporting of eGFR increased nephrology referral volume, patient waiting times, and nephrologists’ workload, without a demonstrable benefit in terms of detection and referral of severe (stage 4 and 5) CKD nor in the reduction of ESRD frequency. Screening programs may need to be implemented along with knowledge translation strategies in order to achievethe goal of preventing and delaying progression of CKD.

Reference: Canadian Society of Nephrology, September 2006. Careand referral of adult patients with reduced kidney function- Position paperfrom the Canadian Society of Nephrology.Available at: http://www.csnscn.ca/local/files/CSN-Documents/CSN%20Postion%20Paper%20Sept2006.pdf
RELATIVE CONTRIBUTIONS OF FIBROSIS AND CONNEXIN CHANGES TOTHE ATRIAL FIBRILLATION SUBSTRATE DURING THE DEVELOPMENT AND REVERSAL OF CONGESTIVEHEART FAILURE
Authors: Burstein Brett , Nishida Kunihiro , Comtois Philippe , Villenuve Louis , Yeh Yung-Hsin , Leung Tack-Ki , Nattel Stanley
Published: May 2017

Background: Connexin alterations occur in various atrial fibrillation (AF) paradigms, but their functional significance remains unclear. No data are available regarding the effects of CHF on atrial connexin expression and phosphorylation. We therefore analyzed connexin changes and their contribution to the AF substrate during the development and reversal ofCHF.

Methods and Results: Dogs were allocated to three groups: CHF induced by 2-week ventricular tachypacing (CHF, n=15); CHF dogs allowed to recover for 4 weeks after 2-week tachypacing (REC, n=15) and non-paced shams (CTL, n=11). Left ventricular end-diastolic pressure increased with CHF (14.5±1.0*** vs.3.7±0.7, ***P < 0.001 vs. CTL) and normalized upon CHF recovery (5.1±1.0^†††, ^††† P < 0.001 vs. CHF). Real-time PCR and Western-blot analyses revealed connexin43 (Cx43) and connexin40 (Cx40) mRNA and protein expression to be unchanged by CHF and REC. However, CHF caused Cx43 dephosphorylation(by ~73%***) and increased Cx40/Cx43 protein ratio (by ~35%***), with both alterations completely reversing in REC. Immunofluorescent confocal microscopy confirmed connexin protein trends, with a reduction in phosphorylated Cx43 (by ~68%*** in CHF) that returned to control in REC. CHF caused conduction abnormalities (phasedelay-range and heterogeneity index, both P < 0.01) and burst pacing-induced AF prolongation (CTL 22±7s, CHF 1100±171s***, REC 884±220s***) which persisted in the recovery period, along with residual fibrosis (CTL 3.6±0.7%, CHF 14.7±1.5%***, REC13.3±2.3%***). Fibrosis physically interrupted muscle bundle continuity and anionically-based action potential model of canine atrium showed that fibrosiswas able to account for the observed conduction abnormalities.

Conclusions: CHF causes connexin-dephosphorylation and Cx40/Cx43ratio increases. With CHF reversal, atrial connexin alterations recover completely, but tissue fibrosis, conduction abnormalities and a substrate forAF remain with fibrosis accounting for conduction abnormalities. Thus, althougha trial connexin changes occur with CHF, they are not essential for conduction disturbances and AF promotion, which appear rather to be related primarily tofibrotic interruption of muscle-bundle continuity.
CONCEPTION AND VALIDATION OF A REFLUX LARYNGITIS MODEL INNEWBORN LAMBS
Authors: Carreau A-M , Patural H , Doueik A , Fortier P-H , Praud J P
Published: May 2017

Background: Daily clinical observations and a recent study (D Vermeylen et al., 2004) suggest that reflux laryngitis (RL) is involved in apneas/ bradycardias of the newborn. The aim of this study is to develop and tovalidate a unique RL model in newborn lambs to test the assumption that a RL provokesan increase in apneas/ bradycardias/ desaturations observed within thelaryngeal chemoreflexes induced by the contact of a liquid with the larynx.

Methods: A surgical instrumentation was performed on the first day of life, which included the insertion of a transcutaneous esophageal catheter in order to allow instillation of a gastric juice surrogate (HCl +pepsin at pH 2) directly on the larynx. The 2 ml-instillations were made 3 times per day for 6 consecutive days. At day 9-10 laryngeal chemoreflexes were induced during sleep. The larynx was removed at the time of necropsy to grade inflammation by a histological study.

Results: Lambs in which a RL was induced (n = 4) presented characteristic signs of RL: cough, weak bleat, reduction in weight gain. Preliminary histological analyses showed a characteristic laryngeal inflammation. Preliminary results showing that apneas and oxygen desaturations seem more prominent in RL lambs than controls provided further validation ofour RL model.

Conclusion: The ovine model developed in this study presents theclinical and histological characteristics of RL observed in newborns. Preliminary results suggesting that a RL increases apneas/bradycardias/desaturations when a liquid is in contact with laryngealmucosa in the newborn must be confirmed.

Supported by: CIHR, FRSQ, Foundation of stars.
STIMULATION OF TOLL-LIKE RECEPTOR 2 BY PLASMODIUMFALCIPARUM GLYCOSYLPHOSPHATIDYLINOSITOLS ENHANCES MACROPHAGE INTERNALIZATION OF PARASITIZED AND UNINFECTED ERYTHROCYTES
Authors: Erdman Laura , Kain Kevin C
Published: May 2017

Background: Toll-like receptors (TLRs) are highly conserved innate sensing receptors that activate host defenses upon detection of microbial products. Plasmodium falciparum glycosylphosphatidylinositols (PfGPI) have been shown to stimulate macrophage cytokine production via TLR2. In addition to their role in inflammation, TLRs may also regulate phagocytosis. P. falciparum parasitized erythrocytes (PEs) can be non-opsonically internalized by macrophages in a process predominantly mediated by scavenger receptor CD36. Moreover, uninfected erythrocytes (UEs) are rendered susceptibleto macrophage clearance during malaria infection due to surface modifications, and this is believed to contribute to the pathogenesis of severe malarialanemia. We hypothesized that stimulation of macrophage TLR2 by PfGPI would enhance innate clearance of PEs as well as malaria-exposed UEs.

Methods: Primaryhuman and murine macrophages were pre-stimulated with PfGPI or asynthetic TLR2 agonist (FSL-1) and phagocytosis assays were performed using P.falciparum PEs, EBABs (PE modelconsisting of anti-CD36 antibodies conjugated to human erythrocytes), IgG-opsonized PEs, and malaria-exposed UEs.

Results: PfGPI and FSL-1 pre-stimulation significantly increased uptake of EBABs and P.falciparum PEs in a TLR2-dependent manner. TLR2 stimulation modestly increased Fc-mediated phagocytosis of IgG-opsonized PEs, and enhanced phagocytosis of UEs isolated from P. falciparum culture.

Conclusion: TLR2-mediated macrophage activation enhanced in vitro clearance of both PEs and malaria-exposed UEs. These data underscore the complexity of TLR involvement in malaria infection: TLR-enhanced phagocytosis may benefit infected individuals by decreasing parasite burden, but in other contexts may predispose to anemia by enhancing UE destruction. Therapeutic targeting of TLR pathways in malaria requires careful consideration.
DISTINCT ROLES FOR DISCOIDIN DOMAIN RECEPTOR 1 (DDR1) EXPRESSED ON BONE MARROW DERIVED CELLS AND VESSEL WALL CELLS DURINGATHEROGENESIS
Authors: Franco C , Hou G , Bendeck M P
Published: May 2017

We have recently described a critical role for the discoidin domain receptor 1 (DDR1) collagen receptor tyrosine kinase in the regulation of fibrosis and inflammation during atherosclerotic plaque development. DDR1 isexpressed on both SMCs and macrophages; however the role of DDR1 expressed in these distinct cells during atherogenesis remains unresolved. In the current study, female Ldlr^-/- mice that were either Ddr1^+/+ or Ddr1^-/-were lethally irradiated and reconstituted with bone marrow from male Ddr1^+/+ or Ddr1^-/- donors yielding three groups of chimeric mice: Ddr1^+/+^?^+/+ (control); Ddr1^+/+^?^-/-^ (vessel wall deletion); and Ddr1^-/-^?^+/+ (bone marrow deletion). Chimeric mice were placed on an atherogenic diet for 12 weeks and hadsimilar body weights, total leukocyte counts, levels of Sry chimerism, and fasting plasma triglycerides at sacrifice, although total cholesterol was increased by 42% in Ddr1^+/+^?^-/- mice. Deletion of DDR1 inbone marrow derived cells (Ddr1^-/-^?^+/+) resulted in a 66% reduction in atherosclerotic lesion area in thedescending aorta compared to Ddr1^+/+^?^+/+ mice. Aortic sinus plaquesfrom Ddr1^-/-^?^+/+ mice were 36% smaller than Ddr1^+/+^?^+/+ plaques but the proportion of plaque area occupied by cells and matrix was similar between groups. By contrast, deletion of DDR1 in vessel wall cells (Ddr1^+/+^?^-/-) resulted in a 57% increase in atherosclerosis in the descending aorta. Furthermore, aortic sinus plaques from Ddr1^+/+ ^?^-/- mice had markedly increased fibrillar collagen and elastin accumulation compared to Ddr1^+/+^?^+/+ plaques resulting in a 156% increase in lesion area and reduced SMC and macrophage content. In conclusion, while DDR1 on bone marrow derived cells is required for plaque development, DDR1 expressed on vessel wall cells negatively regulates plaque matrix accumulation and results in the formation of larger lesions with altered cellular composition. Our data suggest a dual role for DDR1 in the regulationof atherogenesis and plaque matrix content.
DEFICIENCY IN PROTEINASE-ACTIVATED RECEPTOR-2 (PAR2) ATTENUATES COLONIC INFLAMMATION IN MICE INDEPENDENT OF BONE MARROW DERIVEDCELLS.
Authors: Hyun Eric , Andrade-Gordon Patricia , Vergnolle Nathalie
Published: May 2017

Background andaims: The role of PAR_2 duringintestinal inflammation is unclear, since its activation in the gut could lead to either pro- or anti-inflammatory properties. The aim of this study was to investigate the effects of PAR_2 deficiency (using PAR_2-deficient mice: PAR_2 ^-/-)in an animal model of colitis and to investigate the role of PAR_2 deficiency on bone marrow-derived cells.

Methods: Colonic inflammation in PAR_2 ^+/+ and PAR_2 ^-/- mice was induced by 2.5 % dextran sodium sulfate(DSS). Chimeric mice (PAR_2^Ch+/+ or PAR_2^Ch-/-) injected with bone marrow cells (BMC) from either PAR_2 ^+/+ or PAR_2 ^-/- mice, were also given DSS.

Results: PAR_2 ^+/+ mice treated with DSS showed a significant increase in leukocyte rolling/adherence, bowel thickness, myeloperoxidase (MPO) activity and macroscopic damage compared to PAR_2 ^-/-. PAR_2^Ch-/- mice, regardless of the source of bone marrow cells injected, showed significantly reduced inflammatory parameters, compared to PAR_2^Ch+/+ injected with PAR_2 ^+/+ bone marrow. A similar degree of DSS-induced inflammation was observed between chimeric PAR_2^Ch+/+ mice injected with either PAR_2 ^-/- or PAR_2 ^+/+bone marrow cells.

Conclusions: Since DSS inflammation was reduced in PAR_2^ch-/- compared with PAR_2^ch+/+ mice, irrespective of the source of donor bone marrow cells, we conclude that PAR_2 expression on recipient tissues rather than on donor bone marrow cells plays a key role in the development and maintenance of DSS-induced colitis. PAR_2 thus appears as an important mediator of colonic inflammation and represents apotential target for the treatment of inflammatory bowel diseases.

Supported by CIHR and the Crohn's and Colitis Foundation of Canada
COMMITTED ADIPOGENIC PROGENITORS ARE RESPONSIBLE FOR NOVEL ADIPOCYTE GENERATION IN RESPONSE TO DIETARY STIMULI
Authors: Joe Aaron , Yi Lin , Vogl A Wayne , Rossi Fabio
Published: May 2017

During adult life white fat depots can expand via hyperplasia and adipocyte infiltration can appear in aging or disease of non-adipose tissues. These observations suggest that stem/progenitor cells exist that can respond to a variety of stimuli to generate new adipocytes. We used a combination of surface markers to prospectively identify a population of cells containing all adipogenic colony-forming potential in skeletal muscle, subcutaneous and perigonadal adipose depots (adipogenic progenitors, APs). We did not observe any generation of cartilage, bone or muscle from AP cultures, suggesting that APs are committed to the adipocyte lineage. Confocal- and immunoelectron- microscopy analysis on tissues from BrdU-treated high fat diet-fed animals confirms the production of new adipocytes during weight gain. APs appear to participate in this response in a depot- specific manner. Furthermore, we observed a significant correlation between the size of the fat pad and frequency of actively cycling AP cells. Taken together, we show that we have identified an abundant population of lineage-restricted adipogenic progenitors. Importantly, our data suggests that during weight gain, new adipose generation relies on proliferation of these cells.

AJ is supported by a Vancouver Coastal Health–CIHR–UBC MD/PhD Studentship Award.
INVESTIGATING THE MOLECULAR AND DEVELOPMENTAL EFFECTS OF VARIOUS CULTURE REGIMES IN A MOUSE MODEL SYSTEM
Authors: Market Brenna A , Zhang Liyue , Magri Lauren S , Golding Michael C , Mann Mellissa RW
Published: May 2017

Background/Purpose: Genomic imprinting is a specialized transcriptional mechanism that results in the unequal expression of alleles based on their parent-of-origin [1]. Many imprinted genes are critical for proper embryonic and fetaldevelopment [2] and disruption of genomic imprinting are associated with many development disorders [3]. Recently, increased frequencies of imprinting disorders have been correlated with the use of assisted reproductive technologies (ARTs)[2]. Rigorous and thorough testing of ARTs is required to determine their influence on genomic imprinting and development. I hypothesize that imprinting maintenance mechanisms are disrupted during early mouse development by the environmental insult of culture media used in human ARTs, and that loss of imprinting correlates with delayed embryonic development.

Methods: The specific aims of my project are to develop a method to evaluate the methylation and expression patterns of 4 known imprinted genes in individual blastocysts.

Results: We have successfully developed a novel method to evaluate both imprinted methylation and expression from a single mouse blastocyst. This method has been tested and results compared to methods used to evaluate imprinted methylation and expression separately; we have determined that results obtained with a combined protocol are equivalent to either alone. I will use this method to evaluate relationships between development rates in culture andgenomic imprinting, as well as the effects of various culture media used formouse and human embryo culture on genomic imprinting.

Conclusion: This analysis allow for a more comprehensive study ofthe effects of environmental insult on genomic imprinting and preimplantation embryo development.

References:

1. Reik W, Walter J. Genomic imprinting:parental influence on the genome. Nat Rev Genet 2001;2:21-32.

2. Rodenhiser D, Mann M. Epigenetics andhuman disease: translating basic biology into clinical applications. CMAJ. 2006;174:341-8.

3.Paoloni-Giacobino A. Epigenetics in reproductive medicine. Pediatr Res 2007;61:51R-57R.
INTERACTION OF CD44 AND HYALURONAN IS THE DOMINANT MECHANISM FOR NEUTROPHIL ADHESION IN INFLAMED LIVER SINUSOIDS
Authors: McDonald Braedon , McAvoy Erin F , Lam Florence , Gill Varinder , Kubes Paul
Published: May 2017

Background: Previous studies have been unable to identify adhesion molecules that mediate neutrophil recruitment within the liver sinusoids. We hypothesise that involved adhesion molecules may represent novel therapeutic targets for combating pathologic liver inflammation.

Methods: Candidate adhesion molecules were identified using a novel in vivo biopanning approach (dual radiolabelled antibody technique) to quantify endothelial expression levels within the liver compared to other organs. Spinning disk intravital microscopy demonstrated the localization of adhesion molecule expression withinthe liver microvasculature. Using knockout mice, bone marrow chimeric mice, and blocking antibodies, candidate adhesion molecules were systematically investigated for a role in neutrophil recruitment in the liver sinusoids of endotoxemic mice using intravital microscopy and in vitro flow chamber assays.

Results: Hyaluronan was identified as disproportionately expressed in the liver versus other organs, and hyaluronan expression was restricted to liver sinusoids. Blocking CD44-hyaluronan interactions reduced neutrophil adhesion in the sinusoids of endotoxemic mice, but had no effect on neutrophil rolling or adhesion in post-sinusoidal venules. Neutrophil but not endothelial CD44 was required for adhesionin sinusoids. Surprisingly, neutrophil CD44 avidity for hyaluronan was not increased in endotoxemia. Instead, activation of CD44-hyaluronan engagement was the result of qualitative modification of hyaluronan by a dramatic induction of serum-derived hyaluronan-associated protein (SHAP) in sinusoids in response to lipopolysaccharide. Lipopolysaccharide-induced hepatic injury was significantly reduced by inhibiting CD44-hyaluronan interactions. Therapeutic administration of anti-CD44 antibody to endotoxemic mice rapidly detached adherent neutrophils and improved sinusoidal perfusion.

Conclusion: These findings reveal CD44 as a potential therapeutic target in systemic inflammatory responses involving liver.
GENDER DIFFERENCES IN BLEEDING PROBLEMS AND IMPLICATIONS FOR THE ASSESSMENT OF A BLEEDING DISORDER
Authors: Pai M , Li Y , Whittaker S , Arnold E , Seecharan J L , Moffat K A , Webert K E , Cook R J , Heddle N , Hayward C PM
Published: May 2017

Background: The value of gender-specific questions in assessing patients for bleeding problems is not well established, nor is the impact of bleeding on sexual and reproductive health.

Methods: We administered the CHAT (clinical history assessment tool) to 256 females and 66 males referred for bleeding problems, and 67 female and 32 male healthy controls. This detailed bleeding history questionnaire included questions about sexual health, blood in semen, and bleeding with circumcision, menses, pregnancies, and childbirth. A diagnosis for each patient was established by independent medical record review. The prevalence of symptoms among patients and controls was compared.

Results: 80% of patients had bleeding disorders (205 women, 54 men). Men with bleeding disorders did not have significantly increased intercourse-related bleeding, bleeding affecting their sex life, or gender-specific bleeding. Women with bleeding disorders had significantly increased intercourse-related bleeding and bleeding affecting their sex life. They also had significantly increased risks of prolonged menses, menses interfering with lifestyle, and menses requiring medical or surgical therapy (all p values < 0.001). Women with bleeding disorders were more concerned about pregnancy and delivery because of bleeding (p=0.0001), and 38% had been told by a doctor not to become pregnant. Yet they did not have increased risks for pregnancy loss or bleeding during pregnancy (p values > 0.1) and had similar numbers of offspring as controls (mean 2.0 vs.1.7).

Conclusion: Gender has an important impact on the manifestations, diagnosis and management of common bleeding disorders. Detailed questions about gender-specific bleeding are useful in assessing women with possible bleeding disorders, as they are at greater risk for bleeding that negatively impacts their sexual and reproductive health.
ETHICAL ANALYSIS IN PUBLIC HEALTH PRACTICE: A MULTI-SECTORAL MIXED-METHODS STUDY
Authors: Upshur Pakes B
Published: May 2017

Rationale: All decisions in public health practice involve implicit value judgements, and many involve explicit reference to ethical principles. Despite the increased awareness, interest and literature in Public Health Ethics in the past decade, there remains little understanding of what public health practitioners or trainees mean by ethics, what meta-ethical foundations shape their approach to ethical dilemmas, and what prior training in ethics they have had or wish to have. This study aims to answer some of these questions and will serve as the basis for the development of resources to aid public health decision-making.

Method: Qualitative and quantitative data were collected from public health practitioners by means of paper and web-based surveys, as well as structured interviews. Data was coded and analysed using SPSS 15.

Results: 16/20 trainees, 70/150 Canadian practitioners, and 508/2058 American practitioners responded to the survey; 10 interviews were conducted. There was remarkable heterogeneity of responses regarding prioritization of values and meta-ethical justification of ethical norms. Respondents reported little training in ethics and considerable in enhancing their skills. Conflict between ethical imperatives and the law were a prominent feature of American, but not Canadian respondents.

Conclusions: Public Health practitioners hold a variety of disparate views regarding ethics in public health. These translate into different understandings of the goals and means of public health, with far reaching implications in all spheres of practice. A Public Health Ethical Reflection tool was developed to enhance ethical awareness in goal setting, planning and implementation of public health interventions.
EVALUATION OF A NOVEL EEG ANALYSIS METHOD WITH POTENTIAL DIAGNOSTIC APPLICATIONS
Authors: Peters S R , Hu B
Published: May 2017

Background: Cerebral cortex oscillations as recorded on electroencephalograms involve multiple frequency bands. Phase locking of oscillations of these different frequencies may provide a mechanism by which regions of the brain communicate efficiently. Differences in the character of such phase locking may potentially be a diagnostic tool to differentiate seizure types, as traditional analysis of clinical EEG recordings has seldom considered phase-clocking as a diagnostic indicator.

Recently, Canolty et al^1 used a novel metric to quantify cross-frequency phase-amplitude coupling during both spontaneous and induced EEG activity. The technique holds advantages over traditional measures, including easy comparison across trials, robustness to amplitude variation, and simple quantification of preferred phase. Traditional analysis of clinical EEG recordings has seldom considered phase-clocking as a diagnostic indicator.

Methods: We adapted the metric of Canolty^1 to perform better with highly rhythmic oscillations, such as those in seizures, by adding multi-segment reshuffling of phase traces. To validate our modified technique, we used artificial sinusoid traces with a known degree of coupling to test the response of our modified analysis method, and to derive empirically, appropriate values for important numerical parameters. Frequency and phase information was acquired with both the Hilbert and wavelet transforms, with similar qualitative results achieved with either.

Results: As an initial exploration of diagnostic potential, we applied our metric to field potential data obtained from an anaesthetized rat preparation. We compared the phase-amplitude coupling profiles of sleep oscillations with those of simulated absence seizures and showed consistent differences in the phase amplitude coupling profiles. The data suggest that such differences may be useful in evaluating human seizure data.

Conclusions: We conclude that our modified method of data analysis provides an effective approach for measuring normalized phase-amplitude coupling in field potential recordings. Future work will aim to evaluate the possible diagnostic uses of phase-amplitude coupling analysis with data from human seizure patients.

Reference: Canolty et al. Science 2006;313:1626.

Supported by CIHR, NSERC, and the Health Research Foundation.
SPECIES-SPECIFIC SEQUENCE VARIATION OF PORCINE ISLET AMYLOID POLYPEPTIDE REDUCES ITS AMYLOIDOGENICITY AND MAY CONTRIBUTE TO IMPROVED FUNCTION AND SURVIVAL OF PIG ISLET XENOGRAFTS
Authors: Potter K , Abedini A , Butterworth S , Driscoll M , Marek P , Baker R , Korbutt G S , Fraser P E , Raleigh D P , Verchere C B
Published: May 2017

Introduction: Long-term success of human islet transplants has been limited. Beta cell mass and function in transplanted islets progressively decline over time, resembling the progressive loss of insulin secretion in type2 diabetes. Transplanted islets, like those in type 2 diabetes, are subject to apoptotic beta cell death and the formation of toxic aggregates of the betacell peptide islet amyloid polypeptide (IAPP). Interestingly, porcine islets consistently maintain long-term normoglycemia when transplanted into immune-deficient, diabetic mice. We hypothesized that transplanted pig islets may not be subject to amyloid formation and amyloid-induced cell death, and that this may contribute to the success of porcine islet grafts.

Methods & Results: Sequencing of porcine IAPP (pIAPP) from neonatal pig islet (NPI)-extracted RNA confirmed notabledifferences from human IAPP (hIAPP), including a proline substitution within the known amyloidogenic region and a glutamine-for-lysine at the N-terminalcleavage site of porcine proIAPP. Fibril formation was assessed by electron miscroscopy (EM) and Thioflavin T fluorescence. While synthetic hIAPP fibrillized within minutes, pIAPP required 6 weeks to form fibrils detectableby EM. Small peptide fragments of pIAPP failed to aggregate. By Thioflavin T fluorescence assay, hIAPP (40 µM) had an average lag time of 1 h and a 1,000-fold increase in fluorescence by 2 h while pIAPP showed little increase in fluorescence after 20 h. Small peptide fragments of pIAPP also failed to aggregate. hIAPP was significantly more toxic to INS-1 cells than pIAPP (20-200 µM), asassessed by TUNEL (16 h) and Alamar blue (24 h). We also failed to detect amyloid in any (0/9) NPI transplanted into streptozotocin diabetic murine recipients, while human islets had extensive amyloid deposition by 8 weeks post-transplant, associated with graft failure.

Conclusion: Porcine IAPP, unlike human IAPP, is only weakly amyloidogenic and cytotoxic. We speculate that decreased fibrillogenicity of porcine compared to human IAPP may underlie the apparent enhanced survival andfunction of pig islet xenografts.

KJP is supported by aChild & Family – Rx & D & CIHR – UBC MD PhD Studentship and a MichaelSmith Foundation for Health Research Junior Trainee Award.