Flavonol and imidazole derivatives block HPV16 E6 activities and reactivate apoptotic pathways in HPV⁺ cells.

High-risk human papillomaviruses (HR-HPVs) cause nearly all cases of cervical cancer, as well as approximately 30% of head and neck cancers. HPV 16 E6, one of two major viral oncogenes, protects cells from apoptosis by binding to and accelerating the degradation of several proteins important in apoptotic signaling, including caspase 8 and p53. We proposed that blocking the interactions between HPV E6 and its partners using small molecules had the potential to re-sensitize HPV(+) cells to apoptosis. To test this idea, we screened libraries of small molecules for candidates that could block E6/caspase 8 binding and identified several candidates from different chemical classes. We tested hits for dose-dependency and specificity in vitro and for toxicity in a cell-based assay and then used this information to select the two best candidates for further testing: myricetin, a flavonol, and spinacine, an imidazole amino-acid derivative of histidine. Both compounds clearly inhibited the ability of E6 to bind in vitro to both caspase 8 and E6AP, the protein that mediates p53 degradation. In addition, both compounds were able to increase the level of caspase 8 and p53 in SiHa cervical cancer cells, resulting in an increase of caspase 3/7 activity. Finally, both myricetin and spinacine sensitized HPV(+) cervical and oral cancer cells, but not HPV(-) cervical and oral cancer cells, to apoptosis induced by the cancer-specific ligand TRAIL, as well as the chemotherapeutic agents doxorubicin and cisplatin. New therapies based on this work may improve treatment for HPV(+) cancer patients.

Salivary proteomics in bisphosphonate-related osteonecrosis of the jaw.

OBJECTIVE: The objective of this study was to identify differentially expressed salivary proteins in bisphosphonate-related osteonecrosis of the jaw (BRONJ) patients that could serve as biomarkers for BRONJ diagnosis. SUBJECTS AND METHODS: Whole saliva obtained from 20 BRONJ patients and 20 controls were pooled within groups. The samples were analyzed using iTRAQ-labeled two-dimensional liquid chromatography-tandem mass spectrometry. RESULTS: Overall, 1340 proteins were identified. Of these, biomarker candidates were selected based on P-value (<0.001), changes in protein expression (≥1.5-fold increase or decrease), and unique peptides identified (≥2). Three comparisons made between BRONJ and control patients identified 200 proteins to be differentially expressed in BRONJ patients. A majority of these proteins were predicted to have a role in drug metabolism and immunological and dermatological diseases. Of all the differentially expressed proteins, we selected metalloproteinase-9 and desmoplakin for further validation. Immunoassays confirmed increased expression of metalloproteinase-9 in individual saliva (P = 0.048) and serum samples (P = 0.05) of BRONJ patients. Desmoplakin was undetectable in saliva. However, desmoplakin levels tended to be lower in BRONJ serum than controls (P = 0.157). CONCLUSIONS: Multiple pathological reactions are involved in BRONJ development. One or more proteins identified by this study may prove to be useful biomarkers for BRONJ diagnosis. The role of metalloproteinase-9 and desmoplakin in BRONJ requires further investigation.

The clinical significance of intrapulmonary vascular dilations in liver transplant candidates.

Intrapulmonary vascular dilations (IPVD) are common in patients with cirrhosis, but the majority do not have hepatopulmonary syndrome (HPS). The clinical significance of IPVD is unknown. Our aim was to determine the clinical impact due to the entire spectrum of IPVD in liver transplant (LT) candidates. A total of 122 evaluees for LT underwent contrast transthoracic echocardiography (cTTE). The degree of shunting was graded 1-3 (severe). HPS was defined as PaO(2) < 70 mmHg in the presence of IPVD and exclusion of other causes of hypoxemia. IPVD were detected in 57/122 (47%), and of these HPS was found in 5. IPVD were associated with higher Alveolar-arterial (A-a) gradients, with the highest occurring in patients with HPS (IPVD vs. no IPVD: p = 0.003; HPS vs. no IPVD: p = 0.004). All patients with HPS had grade 3 shunting, and had significantly widened A-a gradient and lower PaO(2) compared with grade 1 or 2 IPVDs. Presence of IPVD did not affect survival measured from evaluation or after LT. Other clinical outcomes were also similar among patients with and without IPVD. IPVD are common among LT candidates. HPS is unlikely in presence of only mild to moderate shunting. Clinical outcomes are similar among patients with and without IPVD.

Human temporomandibular joint and myofascial pain biochemical profiles: a case-control study.

Neurobiological mechanisms of human musculoskeletal pain are poorly understood. This case-control study tested the hypothesis that biomarkers within temporomandibular muscle and joint disorders (TMJD) subjects' masseter muscles or temporomandibular joint (TMJ) synovial fluid correlate with plasma biomarker concentrations. Fifty subjects were recruited and categorized into TMJD cases (n=23) and pain-free controls (n=27) at the University of Minnesota School of Dentistry. Prior to specimen collection, pain intensity and pressure pain threshold masseter muscles and the TMJs were assessed. We collected venous blood; biopsied masseter muscle; and sampled TMJ synovial fluid on the subjects' side of maximum pain intensity. We assayed these tissues for the presence of nerve growth factor (NGF), bradykinin (BK), leukotreine B(4) (LTB(4) ) and prostaglandin E(2) (PGE(2) ), F(2) -isoprostane (F(2) I) and substance P (SP). The data was analyzed using Spearman Correlation Coefficients. We found that only plasma concentrations of bradykinin statistically correlated with synovial fluid concentrations (ρ=-0·48, P=0·005), but no association was found between pain intensities. The data suggests that biomarkers used to assess TMJD need to be acquired in a site-specific manner. We also discovered that F(2) I concentrations were associated with muscle pain intensity and muscle pressure pain threshold (PTT) (ß=0·4, 95%CI: 0·03-0·8) and joint PPT (ß=0·4, 95%CI: 0·07-0·8) suggesting that muscle oxidative stress is involved in myofascial pain and that F(2) -I may be a biomarker for myofascial pain.

The range of the Oxford Shoulder Score in the asymptomatic population: a marker for post-operative improvement.

INTRODUCTION: The Oxford Shoulder Score (OSS) is a validated scoring system used to assess the degree of pain and disability caused by shoulder pathology. To date there is no knowledge of the range of the OSS in the healthy adult population. This study aimed to establish the range in asymptomatic individuals. METHODS: The OSS of 100 asymptomatic volunteers was compared with the pre-operative OSS of 100 symptomatic individuals who had had elective shoulder surgery performed at the Royal Preston hospital. RESULTS: The difference in mean scores in the operated group (36.7) and the asymptomatic group (15.3) was statistically significant (p<0.0001). There was, however, a substantial overlap between the scores of the two groups (operated group range: 19-55, asymptomatic group range: 12-47). Factors such as age, sex, body mass index, co-morbidities and smoking did not have a statistically significant impact on the eventual score in the asymptomatic group. CONCLUSIONS: This study has established the range of OSS in the asymptomatic adult population. Symptom scores can only be used effectively when the range in the asymptomatic population is known. This is so that disease severity can be gauged in the context of the normal population and post-operative improvements can be forecast more accurately.

Septic arthritis or transient synovitis of the hip in children: the value of clinical prediction algorithms.

The crucial differentiation between septic arthritis and transient synovitis of the hip in children can be difficult. In 1999, Kocher et al introduced four clinical predictors which were highly predictive (99.6%) of septic arthritis. These included fever (temperature > or = 38.5 degrees C), inability to bear weight, white blood-cell count > 12.0 x 10(9) cells/L and ESR > or = 40 mm/hr; CRP > or = 20 mg/L was later added as a fifth predictor. We retrospectively evaluated these predictors to differentiate septic arthritis from transient synovitis of the hip in children over a four-year period in a primary referral general hospital. When all five were positive, the predicted probability of septic arthritis in this study was only 59.9%, with fever being the best predictor. When applied to low-prevalence diseases, even highly specific tests yield a high number of false positives and the predictive value is thereby diminished. Clinical predictors should be applied with caution when assessing a child with an irritable hip, and a high index of suspicion, and close observation of patients at risk should be maintained.

Arthrosopic knot pushers. Does one size fit all?

Little is known about how knot-pusher design affects arthroscopic knot tying. In our practice, we observed the knot-pusher riding onto the arthroscopic knot at the point of maximum tightening. This can lead to snagging of the knot, which is undesirable as it may lead to loosening of, or damage to the knot. The aim of this study is to determine the optimum size of a knot-pusher to efficiently push the knot without overriding or snagging it. We used an apparatus to model arthropcopic knot tying. Ten examples each of the Duncan loop were tied under controlled conditions of load using one polydioxanone (PDS) monofilament absorbable suture (Ethicon, Livingston, UK), two Ethibond, two Fibrewire and two Panacryl. The loop of the knot was then secured and a 50 N force applied to tension the knot. The suture diameter was measured. Then the knot diameter was measured in two planes using an analogue micrometer. The internal diameter of a Mitek knot-pusher was measured. The mean maximum diameter for each knot was respectively PDS, 2.061 +/- 0.13 mm; Panacryl, 1.907 +/- 0.14 mm; Ethibond, 1.717 +/- 0.16 mm and Fibrewire, 1.654 +/- 0.14 mm. There were significant differences in size between knots tied with different materials except between Ethibond and Fibrewire where the difference was not significant. For each set of knots the smallest maximum knot diameter observed was identified. This was respectively PDS, 1.92 mm; Ethibond, 1.476 mm; Fibrewire, 1.488 mm and Panacryl, 1.715 mm. The internal diameter of a Mitek knot-pusher was found to be 1.95 mm. The current Mitek knot-pusher appears to be well suited to one PDS and two Panacryl. It appears less ideal for two Ethibond and two Fibrewire. One knot-pusher does not fit all and we suggest that different knot-pushers be used for different suture materials.

Modulation of apoptosis by human papillomavirus (HPV) oncoproteins.

The regulation of host-mediated apoptosis by the E6 and E7 oncoproteins has garnered attention because it is believed to be an important strategy employed by high-risk (HR)-human papillomaviruses (HPVs) to evade immune surveillance. Additionally, the revelation that E5 can protect cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis suggests that it may also play a role in undermining host defense mechanisms. Cellular transformation is an unintended consequence of persistent infection by HR-HPVs, and it is therefore likely that the primary function of E5, E6 and E7 is to regulate cell survival throughout the normal viral life cycle in order to ensure viral replication and promote the spread of progeny. The purpose of this article is to review the literature on the regulation of host-mediated apoptosis by E5, E6 and E7 that describes the mechanisms employed by HR-HPVs to persist in the host and create the conditions necessary for cellular transformation.

Accelerated degradation of FADD and procaspase 8 in cells expressing human papilloma virus 16 E6 impairs TRAIL-mediated apoptosis.

Viruses have developed sophisticated strategies to evade host defenses and facilitate the production and spread of progeny. In this study, we show that transfection of the human papillomavirus (HPV) 16 E6 oncogene into HCT116 cells provides protection from tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Additionally, we demonstrate that the protection provided by E6 is dose-dependent because higher levels of E6 provide greater protection. The mechanism underlying this protection involves a rapid reduction in the protein levels of both Fas-associated death domain (FADD) and procaspase 8, which results in suppression of the activation of caspases 8, 3 and 2. Interestingly, E6 does not interfere with the mitochondrial apoptotic pathway even though HCT116 cells have been classified as type II cells with regard to TRAIL signaling. These findings demonstrate that E6 has a more generalized effect on signaling by death ligands than was previously thought and support the notion that E6 can utilize p53-independent mechanisms to modulate cell survival.

The human papillomavirus 16 E6 protein can either protect or further sensitize cells to TNF: effect of dose.

High-risk strains of human papillomavirus, including HPV 16, cause human cervical carcinomas, due in part to the activity of their E6 oncogene. E6 interacts with a number of cellular proteins involved in host-initiated apoptotic responses. Paradoxically, literature reports show that E6 can both protect cells from and sensitize cells to tumor necrosis factor (TNF). To examine this apparent contradiction, E6 was transfected into U2OS cells and stable clones were treated with TNF. Intriguingly, clones with a high level of E6 expression displayed an increased sensitivity to TNF by undergoing apoptosis, while those with low expression were resistant. Furthermore, TNF treatment of cells in which the expression of E6 was regulated by the addition of doxycycline demonstrated clearly that while low levels of E6 protect cells from TNF, high levels sensitize cells. Together, these results demonstrate that virus-host interactions can be complex and that both quantitative and qualitative aspects are important in determining outcome.

Botulinum toxin injection in the treatment of tennis elbow. A double-blind, randomized, controlled, pilot study.

BACKGROUND: A recent report has suggested that local injection of botulinum toxin type A is an effective method of treatment for chronic tennis elbow. The toxin is thought to provide temporary paralysis of the painful common extensor origin, thereby allowing a healing response to occur. To test this theory, we performed a double-blind, randomized, controlled, pilot trial comparing injections of botulinum toxin type A with those of a placebo (normal saline solution) in the treatment of chronic tennis elbow. METHODS: Forty patients with a history of chronic tennis elbow for which all conservative treatment measures, including steroid injection, had failed were randomized into two groups. Half the patients received 50 units of botulinum toxin type A, and the remainder received normal saline solution. The intramuscular injections were performed 5 cm distal to the maximum point of tenderness at the lateral epicondyle, in line with the middle of the wrist. The two solutions used for the injections were identical in appearance and temperature. The results of a quality-of-life assessment with the Short Form-12 (SF-12), the pain score on a visual analogue scale, and the grip strength measured with a validated Jamar dynamometer were recorded before and three months after the injection. RESULTS: Three months following the injections, there was no significant difference between the two groups with regard to grip strength, pain, or quality of life. CONCLUSIONS: With the numbers studied, we failed to find a significant difference between the two groups; thus, we have no evidence of a benefit from botulinum toxin injection in the treatment of chronic tennis elbow.

Boundary conditions at the tendon-bone interface.

The reconstruction of a tendon-bone interface, as in rotator-cuff repairs, remains a challenging surgical problem. There is however, little data to show what effect joint position or repair loading under physiological conditions have on the repaired tendon-bone interface. A change in the amount of contact area or load at the tendon-bone interface may influence healing. In this study we investigated the effect of limb position and boundary conditions on the tendon-bone interface in an in vitro rabbit tendon-bone repair model using both unlinked and linked suture repairs.

Phytoestrogens are potent inhibitors of estrogen sulfation: implications for breast cancer risk and treatment.

We investigated the ability of 37 flavonoids and flavonoid sulfoconjugates, including some abundant dietary constituents, to act as substrates and/or inhibitors of the sulfotransferase and sulfatase enzymes that interconvert active estrogens and inactive estrogen sulfates in human tissues. The enzymes studied include estrogen sulfotransferase, the thermostable phenolsulfotransferase that acts on a range of substrates including estrogens; steroid sulfatase; and two related enzymes, monoamine phenolsulfotransferase and arylsulfatase A. Several dietary flavonoids, including the soy isoflavones genistein and daidzein, were sulfated by these human sulfotransferases. Many flavonoids were potent inhibitors of thermostable phenolsulfotransferase. Genistein and equol were potent mixed inhibitors of hepatic estrogen sulfotransferase, with inhibitory constant values of 500 nM and 400 nM, respectively. Monoamine phenolsulfotransferase activity was relatively unaffected by flavonoids, but this enzyme was mainly responsible for the sulfation of flavonoids at concentrations greater than 1 micro M. Of the compounds tested, only daidzein 4,7-bisulfate, a trace metabolite in humans, significantly inhibited steroid sulfatase in the micromolar concentration range. Hence, dietary flavonoids may be able to influence the bioavailability of endogenous estrogens, and disrupt endocrine balance, by increasing the ratio of active estrogens to inactive estrogen sulfates in human tissues.

The kinematics and kinetics of slipknots for arthroscopic Bankart repair.

An apparatus has been developed to enable the in vitro measurement of the kinematics and kinetics of a slipknot, both while the knot is being tightened and with the knot in place and the tension removed. During tightening, the apparatus provides a linearly increasing resistance, which may be considered analogous to the resistance experienced when the labrum is drawn toward and apposed to the edge of the glenoid cavity during a Bankart repair. The tension to close the knot is measured with a tensiometer, in tandem with the closure of the capsule model onto the bone anchor, which is measured with a datalogger. The tightening tension was limited to 25 N and intraoperator and interoperator comparisons were made for four knots tied by six participants. Failure of a knot was taken to be reverse slippage of 2 mm. A second criterion of ease of slide was used to assess each knot. We found wide interoperator variability with regard to slipknot tying. A methodology for quality control of slipknot tying is presented.

Acute transverse myelitis and Guillain-Barré overlap syndrome with serological evidence for mumps viraemia.

Both acute transverse myelitis (ATM) and Guillain-Barré syndrome (GBS) occur as rare associations with mumps viraemia but to our knowledge, concurrent ATM and GBS related to mumps has only been reported once previously. We describe the case of a young woman presenting with confusion and collapse 2 weeks after a flu-like illness. An initial diagnosis of transverse myelitis was made on the basis of the clinical findings and radiological evidence of a swollen spinal cord with uniform high signal change on T2 weighted MRI. The patient was treated with intravenous methylprednisolone without significant recovery. The diagnosis was later revised to include GBS on the basis of worsening facial diplegia in the setting of a flaccid tetraparesis, and neurophysiological evidence of a sensorimotor axonal polyradiculoneuropathy. Acute mumps viraemia was confirmed on serological grounds. The patient made an improvement in ventilatory capacity with intravenous immunoglobulin treatment.

Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry.

The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.

Do dietary phytoestrogens influence susceptibility to hormone-dependent cancer by disrupting the metabolism of endogenous oestrogens?

Phytoestrogens are natural constituents of our diets that have been suggested to protect against hormone-dependent breast cancer. Some of the diverse effects of these compounds may be attributed to ligand-dependent differences in their interaction with oestrogen receptor sub-classes. However, phytoestrogens can also inhibit enzymes that are involved in the generation and removal of endogenous steroid hormones. Among the most potent effects of dietary phytoestrogens is their ability to inhibit the sulphotransferases that sulphate both oestrogenic steroids and a variety of environmental chemicals, including dietary pro-carcinogens. Circulating steroid sulphates are thought to be the major source of oestradiol in post-menopausal breast tumours and sulphation is a key step in the activation of some dietary pro-carcinogens. Hence the inhibition of sulphotransferases by dietary phytoestrogens may have complex effects upon human susceptibility to breast cancer.

Activation of a p53-independent, sphingolipid-mediated cytolytic pathway in p53-negative mouse fibroblast cells treated with N-methyl-N-nitro-N-nitrosoguanidine.

Sphingolipids such as ceramide are important mediators of apoptosis and growth arrest triggered by ligands such as tumor necrosis factor and Fas-L binding to their receptors. When LM (expressing p53) and LME6 (lacking p53) cells were exposed to the genotoxin N-methyl-N-nitro-N-nitrosoguanidine (MNNG), both cell lines underwent cytolysis in a very similar manner, suggesting the presence of a p53-independent apoptotic response to this genotoxic stress. To determine whether sphingolipids such as ceramide might serve as mediators in this system, the responses of these cells to exogenous sphingolipids as well as their changes in endogenous sphingolipid levels after DNA damage were examined. Treatment with exogenous C2-ceramide and sphingosine led to cell death in both LM and LME6, and treatment of the LME6 cells with MNNG resulted in a transient increase in intracellular ceramide of approximately 50% over a period of 3 h. Finally, treatment with the de novo inhibitor of ceramide synthesis ISP-1 protected LME6 cells from MNNG-triggered cell death. This MNNG-triggered induction of ceramide was not observed in the p53-expressing LM cells, suggesting that it may be down-regulated by p53. Although ceramide-mediated cell death can proceed in the absence of p53, exogenously added C2-ceramide increased the cellular p53 level in LM cells, suggesting that the two pathways do interact.