Tachykinin receptor modulation of cyclooxygenase-2 expression in human polymorphonuclear leucocytes

Background and purpose:

We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs).

Experimental approach:

The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E₂ (PGE₂) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP.

Key results:

Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol·L⁻¹-µmol·L⁻¹ concentration range, modulated COX-2 expression and PGE₂ release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar⁹, Met(O₂)¹¹]SP, [β-Ala⁸] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-κB concentration-dependently, with a maximum effect at 1 nmol·L⁻¹.

Conclusions and implications:

Human PMNs possess functional NK₁, NK₂ and NK₃ receptors, which mediate the induction of COX-2 expression and NF-κB activation by SP.     

Cholesterol as a determinant of cooperativity in the M2 muscarinic cholinergic receptor

M2 muscarinic receptor extracted from Sf9 cells in cholate-NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[3H]methylscopolamine (NMS) is only 50% of that for [3H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [3H]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588-604]. In contrast, M2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [3H]NMS at either 30 or 4 degrees C, although there was a time-dependent inactivation during incubation with [3H]NMS at 30 degrees C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol-methyl-beta-cyclodextrin. M2 receptors extracted from treated Sf9 membranes were stable at 30 and 4 degrees C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M2 muscarinic receptor.     

Raloxifene, tamoxifen and vascular tone

1. Oestrogen deficiency causes progressive reduction in endothelial function. Despite the benefits of hormone-replacement therapy (HRT) evident in earlier epidemiological studies, recent randomized trials of HRT for the prevention of heart disease found no overall benefit. Instead, HRT users had higher incidences of stroke and heart attack. Most women discontinue HRT because of its many side-effects and/or the increased risk of breast and uterine cancer. This has contributed to the development of selective oestrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, as alternative oestrogenic agents. 2. A SERM is a molecule that binds with high affinity to oestrogen receptors but has tissue-specific effects distinct from oestrogen, acting as an oestrogen agonist in some tissues and as an antagonist in others. Clinical and animal studies suggest multiple cardiovascular effects of SERMs. For example, raloxifene lowers serum levels of cholesterol and homocysteine, attenuates oxidation of low-density lipoprotein, inhibits endothelial-leucocyte interaction, improves endothelial function and reduces vascular smooth muscle tone. 3. Available evidence suggests that raloxifene and tamoxifen are capable of acting directly on both endothelial cells and the underlying vascular smooth muscle cells and cause a multitude of favourable modifications of the vascular wall, which jointly contribute to improved local blood flow. The outcome of the Raloxifene Use for the Heart (RUTH) trial will determine whether raloxifene, currently approved for the treatment of post-menopausal osteoporosis, could substitute for HRT in alleviating cardiovascular symptoms in post-menopausal women.     

DENTAL CARIES AND PERIODONTAL DISORDERS IN CHRONIC LIVER DISEASE

Bacterial infections are frequent complications in patients with chronic liver disease (CLD). A potential source of infection may be dental foci. This study was carried out to assess the association of CLD with dental caries and periodontal disease. Dental caries and periodontal examinations were performed prospectively in patients with CLD (group A) and controls without any liver disease (group B). Similar examination was also carried out in alcoholics without liver disease (group C) as well as in cases with portal hypertension but no liver disease (group D) i.e. patients with Non Cirrhotic Portal Fibrosis and Extrahepatic portal obstruction. A total of 231 subjects (Group A:83, group B: 75, group C:46 and group D:27) were studied. Group A included 32 cases with chronic hepatitis B&C, 26 with alcoholic cirrhosis, 14 with postnecrotic cirrhosis, and 11 with cryptogenic cirrhosis. Measures of oral hygiene (p < 0.01), dental care (p < 0.001), and periodontal parameters were worse and the number of teeth requiring treatment (p < 0.05) was higher in alcoholics with or without cirrhosis than in healthy subjects and nonalcoholic patients with cirrhosis. Alcoholics had a lower, total number of teeth than patients without alcohol abuse and healthy controls (p < 0.01). The dental caries and periodontal status of patients with nonalcoholic cirrhosis did not differ significantly from group B. The severity and duration of liver disease had no influence on dental caries and periodontal disease. The presence of chronic alcohol abuse rather than cirrhosis or portal hypertension is a major predisposing factor for dental caries and periodontal diseases. In alcoholics, these diseases appear to be caused primarily by bad oral hygiene and poor dental care.KEY WORDS: Alcohol abuse, Chronic liver disease, Dental caries, Periodontal disease     

Spatial and temporal analysis of nociception-related spinal cord matrix metalloproteinase expression in a murine neuropathic pain model

BackgroundAlthough the mammalian central nervous system contains numerous matrix metalloproteinases (MMPs), the significance of each MMP relative to nociception remains obscure. Working from the hypothesis that MMPs may be involved in activity-dependent reorganization during neuronal modulation, we explored the role of each MMP following neuropathological injury by establishing MMP expression profiles in a murine model for neuropathic pain.MethodsSciatic nerves of adult male C57BL/6C mice were partially ligated, and their responses to mechanical and radiant heat stimulations were observed at 1, 3, 7, and 14 days. The expression of several nociception-related MMPs (MMP-2, MMP-9, MMP-12, MMP-17, and MMP-24) in the spinal cord was detected by immunohistochemical analysis, Western blotting, and real-time polymerase chain reaction. In addition, the potential of GM6001, a general inhibitor of MMP peritoneal administration, to modulate nociceptive pain responses in a chronic neuropathic pain model in mice was also investigated.ResultsMMP-2, 9, 17, and 24, but not MMP-12, were expressed in the murine spinal cord. MMP-9 was constitutively expressed in neurons and microglial cells, immediately upregulated after nerve injury, and returned to baseline levels at day 3. Expression of MMP-2, MMP-17, and MMP-24 gradually increased after nerve injury. Morphologically, MMP-2-positive cells were glial-like cells. MMP-17 and MMP-24 expression was widespread in gray matter, neurons, and microglial cells, and concentrated in the marginal zone of the dorsal horn and in small capillaries. Peritoneal administration of GM6001 resulted in significantly attenuated thermal hyperalgesia and tactile allodynia induced by nerve injury.ConclusionExpression of several nociception-related MMPs was differentially regulated both temporally and spatially following nerve injury. These results suggest that neuronal remodeling requires concerted expression of particular MMPs in specific temporal and spatial patterns, which may be necessary for neuronal plasticity and/or recovery.     

Human Papillomavirus DNA Detection in Menstrual Blood from Patients with Cervical Intraepithelial Neoplasia and Condyloma Acuminatum

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women''s menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases.Cervical cancer (CC) is the second most common malignancy and cause of cancer-related death in women worldwide (17). The well-defined premalignant phase of this cancer has contributed to the success of the cytology-based Papanicolaou (Pap) screening test; the incidence of CC in resource-rich countries has been dramatically reduced by the use of this test (11). However, the Pap test has several limitations: (i) the collection of cervical cells can create discomfort and embarrassment (7); (ii) evaluation of test results in cytology screening involves subjective assessments with high susceptibility to intraindividual and interindividual variability (11); (iii) the test exhibits low (around 51%) sensitivity for detection of high-grade (HG) cervical intraepithelial neoplasia (HGCIN) (11); and (iv) cytology-trained personnel and special equipment with excellent quality control are needed, which involves a huge amount of resources (3). Therefore, there is a need for another cervical screening test that can overcome these limitations. Recent studies have shown that the human papillomavirus (HPV) DNA test is more sensitive than the Pap test in detecting HGCIN (11-13). However, the sample collection method, using a cytobrush, is still invasive and unpleasant, which would affect a woman''s decision whether to take the Pap test. We hypothesized that menstrual blood (MB) collected in sanitary napkins would contain HPV-infected cervical cells from patients with CIN or condyloma acuminatum (CAC), as HPV is known to be the etiological agent for both conditions (10). In this study, we explored the possibility of detecting and typing HPV DNA in MB collected in sanitary napkins from patients with CIN or CAC. As a high HPV16 DNA load has been reported to be associated with a higher grade of CIN or a higher risk of development of HGCIN during follow-up (5, 19), it would be interesting to explore whether HPV16 DNA loads can be measured using quantitative real-time PCR (QRT-PCR) with MB specimens. The information obtained would be important for enhancing understanding of the diagnostic and prognostic potential of MB HPV DNA for patients with CIN and CAC, and possibly for determining whether MB HPV DNA can be used as the basis for a noninvasive screening test for detection of these premalignant cervical diseases (PCD).     

A splice‐site mutation leads to haploinsufficiency of EXT2 mRNA for a dominant trait in a large family with multiple osteochondromas

Multiple osteochondromas (MO) is an autosomal‐dominant disorder and mutations in EXT1 and EXT2 account up to 78% of the cases studied, including missense, nonsense, frameshift, and splice‐site mutations. EXT1 and EXT2 encode glycosyltransferases required for the synthesis of heparan sulfate (HS) chains. The molecular pathogenesis underlying these mutations is still largely unknown. A heterozygous c.1173 + 1G > T (EXT2) mutation was identified in a three‐generation 34‐member MO family and is present in all 19 affected members. The consequence of this mutation is exon 7 being spliced out, and the result is a shift in the codon‐reading frame from position 360 (R360) of the amino acid sequence leading to a premature termination codon, and the mutant mRNA is degraded to an undetectable level. Interestingly, HS glycosaminoglycans were also undetectable in the cartilage cap of the tumors by immunostaining. Full penetrance of this mutation in all affected members ranging from 5 to 70 years of age suggests this primary defect in EXT2 mRNA level, in conjunction with other cellular changes such as enhanced heparanase expression, can produce profound effect on the synthesis of HS chains in cartilage, the consequence of which impacts on the regulation of chondrocyte proliferation and differentiation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1522–1530, 2010     

Gadolinium-enhanced cardiovascular magnetic resonance: administered dose in relationship to united states food and drug administration (FDA) guidelines

Purpose

Myocardial late gadolinium enhancement was originally validated using higher than label-recommended doses of gadolinium chelate. The objective of this study was to evaluate available evidence for various gadolinium dosing regimens used for CMR. The relationship of gadolinium dose warnings (due to nephrogenic systemic fibrosis) announced in 2008 to gadolinium dosing regimens was also examined.

Methods

We conducted a meta-analysis of peer reviewed publications from January, 2004 to December, 2010. Major subject search headings (MeSh) terms from the National Library of Medicine''s PubMed were: contrast media, gadolinium, heart, magnetic resonance imaging; searches were limited to human studies with abstracts published in English. Case reports, review articles, editorials, MRA related papers and all reports that did not indicate gadolinium type or weight-based dose were excluded. For all included references, full text was available to determine the total administered gadolinium dose on a per kg basis. Average and median dose values were weighted by the number of subjects in each study.

Results

399 publications were identified in PubMed; 233 studies matched the inclusion criteria, encompassing 19,934 patients with mean age 54.2 ± 11.4 (range 9.3 to 76 years). 34 trials were related to perfusion testing and 199 to myocardial late gadolinium enhancement. In 2004, the weighted-median and weighted-mean contrast dose were 0.15 and 0.16 ± 0.06 mmol/kg, respectively. Median contrast doses for 2005-2010 were: 0.2 mmol/kg for all years, respectively. Mean contrast doses for the years 2005-2010 were: 0.19 ± 0.03, 0.18 ± 0.04, 0.18 ± 0.10, 0.18 ± 0.03, 0.18 ± 0.04 and 0.18 ± 0.04 mmol/kg, respectively (p for trend, NS). Gadopentetate dimeglumine was the most frequent gadolinium type [114 (48.9%) studies]. No change in mean gadolinium dose was present before, versus after the Food and Drug Administration (FDA) black box warning (p > 0.05). Three multi-center dose ranging trials have been published for cardiac MRI applications.

Conclusion

CMR studies in the peer-reviewed published literature routinely use higher gadolinium doses than regulatory agencies indicated in the package leaflet. Clinical trials should be supported to determine the appropriate doses of gadolinium for CMR studies.