BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel.

The incidence of melanoma has increased rapidly over the past 30 years, and the disease is now the sixth most common cancer among men and women in the U.K. Many patients are diagnosed with or develop metastatic disease, and survival is substantially reduced in these patients. Mutations in the BRAF gene have been identified as key drivers of melanoma cells and are found in around 50% of cutaneous melanomas. Vemurafenib (Zelboraf(®) ; Roche Molecular Systems Inc., Pleasanton, CA, U.S.A.) is the first licensed inhibitor of mutated BRAF, and offers a new first-line option for patients with unresectable or metastatic melanoma who harbour BRAF mutations. Vemurafenib was developed in conjunction with a companion diagnostic, the cobas(®) 4800 BRAF V600 Mutation Test. The purpose of this paper is to make evidence-based recommendations to facilitate the implementation of BRAF mutation testing and targeted therapy in patients with metastatic melanoma in the U.K. The recommendations are the result of a meeting of an expert panel and have been reviewed by melanoma specialists and representatives of the National Cancer Research Network Clinical Study Group on behalf of the wider melanoma community. This article is intended to be a starting point for practical advice and recommendations, which will no doubt be updated as we gain further experience in personalizing therapy for patients with melanoma.

Studies on the mechanism of bacterial resistance to complement-mediated killing. II. C8 and C9 release C5b67 from the surface of Salmonella minnesota S218 because the terminal complex does not insert into the bacterial outer membrane.

The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8- deficient serum and washed to remove al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of less than 1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.

Role of the gamma chain Ala-Gly-Asp-Val and Aalpha chain Arg-Gly-Asp-Ser sites of fibrinogen in coaggregation of platelets and fibrinogen-coated beads.

Fibrinogen (Fg) mediates platelet aggregation and adhesion to artificial surfaces. The carboxyl terminus of the gamma chain of Fg (residues AGDV at gamma408-411) is known to play an exclusive role in platelet aggregation, while there is no known role for the consensus RGD sites in the Aalpha chain. In this study, we used flow cytometry to measure the coaggregation (CA) of platelets with Fg-coated beads, and investigated which domains in surface-immobilized Fg support platelet adhesion. CA of platelets with Fg-beads was nearly abolished in the presence of 4A5, a monoclonal antibody (mAb) whose epitope includes AGDV, while Z69/8, a mAb that also binds to the gamma chain carboxyl terminus but does not cover AGDV, had little effect. When beads were coated with recombinant Fg (rFg) lacking AGDV, CA was similarly abolished. In contrast, beads coated with Fg that lacked the RGDS site, supported platelet CA as did intact Fg. These results were confirmed in experiments that measured the binding of activated soluble glycoprotein IIb and IIIa (GPIIbIIIa), the platelet membrane glycoprotein complex known to be the Fg receptor, to immobilized Fg. This binding was inhibited by mAb 4A5, but not by mAb Z69/8. Binding was totally retained when beads were coated with Fg lacking RGDS, but was completely lost when beads were coated with Fg lacking AGDV. These results demonstrated that the AGDV sequence on the carboxyl terminus of the gamma chain of Fg plays an exclusive role in platelet adhesion to surface-immobilized Fg, while the carboxyl terminus of the Aalpha chain, including a consensus RGD site, is not required.

Calcium-induced condensation-reorganization phenomena in multilamellar vesicles of phosphatidic acid. pH potentiometric and 31P-NMR, Raman and ESR spectroscopic studies.

In biological membranes, the anionic characteristics of the polar headgroup of phosphatidic acids are responsible for structural changes induced by Ca2+ in many cellular processes. The very simple headgroup structure of dipalmitoylphosphatidic acid (DPPA) offers particular advantages as a model to study the interactions between Ca2+ and natural phosphatidic acids such as cardiolipin and phosphatidylserine. The effects of calcium ions on DPPA membranes have been studied as a function of temperature by potentiometry and by Raman, ESR and 31P-NMR spectroscopies. The protons in monosodic DPPA liposomes have been considered as a probe to detect pH variations resulting from introduction of Ca2+ inside the membrane. This method has also allowed us to determine the stoichiometry of this reaction: 2 DPPA(H) + Ca2+----Ca(DPPA)2 + 2H+. 31P-NMR spectroscopy has been used to detect reorganization-condensation phenomena in multilamellar vesicles of DPPA under the influence of calcium and temperature. Furthermore, the temperature profiles obtained from Raman spectra for Ca(DPPA)2 membranes provide conclusive evidence that Ca2+ induces major reorganization of the phosphatidic acid component into a highly ordered phase. Quantitative estimates of the degree of motional restriction of spin-labeled soaps embedded inside membranes composed of DPPA with or without Ca2+ have been made using ESR technique. These results are discussed and compared to those found previously for a natural phosphatidic acids such as phosphatidylserine.

Antitumor efficacy, pharmacokinetics, and biodistribution of NX 211: a low-clearance liposomal formulation of lurtotecan.

Lurtotecan is a clinically active water-soluble camptothecin analogue that has been formulated into a low-clearance unilamellar liposome, NX 211. Comparative studies between free drug and NX 211 have been performed assessing pharmacokinetics in nude mice, tissue distribution in tumor-bearing mice, and antitumor efficacy in xenografts. Compared with lurtotecan, NX 211 demonstrated a significant increase in plasma residence time and a subsequent 1500-fold increase in the plasma area under the drug concentration curve. The volume of distribution was also greatly restricted, suggesting altered tissue distribution. Evaluation of tissues 24 h after administration of either [14C]NX 211 or [14C]lurtotecan to ES-2 tumor-bearing mice demonstrated a 40-fold increase in radiolabeled compound in the tumors of NX 211-treated mice compared with mice treated with lurtotecan. In single-dose efficacy studies, NX 211 produced a consistent 3-fold or greater increase in therapeutic index compared with lurtotecan in both the KB and ES-2 xenograft models. When compared at equitoxic levels in repeat-dose efficacy studies, NX 211 generated durable cures lasting >60 days and a 2-8-fold increase in log10 cell kill, compared with lurtotecan and topotecan, respectively. Together, these data demonstrate that NX 211 has significant therapeutic advantage over lurtotecan and that the improved antitumor activity is consistent with increased exposure and enhanced drug delivery to tumor sites.

Vibrational spectroscopic and ultrasound analysis for in-process characterization of high-density polyethylene/polypropylene blends during melt extrusion.

Spectroscopic techniques such as Raman, mid-infrared (MIR), and near-infrared (NIR) have become indispensable analytical tools for rapid chemical quality control and process monitoring. This paper presents the application of in-line Fourier transform near-infrared (FT-NIR) spectroscopy, Raman spectroscopy, and ultrasound transit time measurements for in-line monitoring of the composition of a series of high-density polyethylene (HDPE)/polypropylene (PP) blends during single-screw extrusion. Melt composition was determined by employing univariate analysis of the ultrasound transit time data and partial least squares (PLS) multivariate analysis of the data from both spectroscopic techniques. Each analytical technique was determined to be highly sensitive to changes in melt composition, allowing accurate prediction of blend content to within +/- 1% w/w (1sigma) during monitoring under fixed extrusion conditions. FT-NIR was determined to be the most sensitive of the three techniques to changes in melt composition. A four-factor PLS model of the NIR blend spectra allowed determination of melt content with a standard prediction error of +/- 0.30% w/w (1sigma). However, the NIR transmission probes employed for analysis were invasive into the melt stream, whereas the single probes adopted for Raman and ultrasound analysis were noninvasive, making these two techniques more versatile. All three measurement techniques were robust to the high temperatures and pressures experienced during melt extrusion, demonstrating each system's suitability for process monitoring and control.

Effects of fluoride on parathyroid hormone secretion and intracellular second messengers in bovine parathyroid cells.

Fluoride ion (F-) alone or in conjunction with aluminum (Al3+) has been shown to stimulate the activity of guanine nucleotide-binding proteins (G proteins) in cell membrane preparations from a variety of cell types and in intact hepatic cells. Several studies have indicated that G proteins are involved in the regulation of parathyroid hormone (PTH) secretion. Intracellular second messengers which modulate PTH secretion (e.g., cAMP) have also been found to be regulated by G proteins. We have, therefore, employed F- as a probe to investigate the possible role of G proteins in the modulation of PTH release and the intracellular second messengers that have been implicated in the control of PTH secretion. F- produces a dose-dependent inhibition of PTH release with a maximal inhibitory effect (67%) at 5 mM. F- exerts its inhibitory effect within 5 min and the degree of suppression of PTH secretion gradually increases over 1 hr. F- (5 mM) inhibits PTH secretion at 0.5 mM Ca2+ to the level observed with 2 mM Ca2+ alone; moreover, the effects of F- and high Ca2+ are not additive. While 1 mM F- suppresses PTH secretion by only 21%, and 10 microM Al3+ has virtually no effect at all, together they inhibit PTH release approximately to the level (63% inhibition) observed with 5 mM NaF alone. In the presence of 10(-5) M dopamine, F- produces a concentration-dependent inhibition of cAMP accumulation (0.684 +/- 0.033 pmoles/10(5) cells at 0 mM F- vs. 0.256 +/- 0.048 at 5 mM F-). However, the F- -induced decrease in cAMP cannot account for the inhibition of PTH release by this agent, since addition of methylisobutylxanthine (10(-4) M) by F- -treated cells raises intracellular cAMP content above that of control cells but fails to reverse the inhibition of PTH release. The cytosolic calcium concentration in Fura-2-loaded cells increases from 210 +/- 20 nM to 340 +/- 44 nM after 5 mM F- was added to incubation media. Prior removal of extracellular Ca2+ by EGTA totally blocks the F- -induced rise in cytosolic Ca2+ without preventing the inhibition of PTH release by NaF. F- also produces a time- and dose-dependent increase in the accumulation of IP, IP2, and IP3 in cells prelabeled with [3H]inositol and incubated with 10 mM Li+, consistent with activation of phospholipase C. We conclude that F- is a potent inhibitor of PTH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of intramembrane particles in proteoliposomes containing yeast mitochondrial ATPase.

Oligomycin-sensitive ATPase was isolated from yeast mitochondria and incorporated into phospholipid vesicles by cholate dialysis. The structure of the membranes was examined by freeze-fracture electron microscopy. Liposomes formed without the ATPase were small with diameters in the range 20 to 40 nm and they had smooth fracture faces. When the ATPase was introduced, larger vesicles formed (up to 500 nm in diameter) which bore intramembrane particles (IMPs) on their fracture faces. The IMP frequency was dependent on the ratio of protein to lipid used in the reconstitution. After unidirectional shadowing at an angle of 45 degrees, IMPs had a mean width of 12.7 nm and a mean shadow length of 5.9 nm. After rotary shadowing, the mean IMP diameter was 12.9 nm. When the amount of protein was higher than 1 mg/10 mg phospholipid in the initial incorporation mixture, the ATPase formed small hexagonal arrays in the liposome membranes. Computer-based image processing of such arrays showed that the IMPs had a centre-to-centre spacing of 13.7 nm. Calculations showed that each IMP may correspond to an oligomer of the ATPase.

Characterization of the dopamine-responsive adenylate cyclase of bovine parathyroid cells and its relationship to parathyroid hormone secretion.

To investigate further the mechanism of dopamine (DA)-stimulated and parathyroid hormone (PTH) secretion, we have identified and studied DA-sensitive adenylate cyclase in a particulate preparation of osmotically lysed dispersed bovine parathyroid cells. Adenylate cyclase was responsive to DA at concentrations as low as 0.3 microM, and the maximal stimulation in the presence of GTP was 2- to 4-fold that of activity with GTP alone. (-)Propranolol (1 microM) abolished the stimulation by (-)isoproterenol but did not inhibit the DA-stimulated adenylate cyclase, whereas alpha-flupenthixol (1 microM) inhibited DA stimulation but not that of (-)isoproterenol. The dopaminergic agonists epinine and 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene were nearly as effective as DA in stimulating the enzyme, while apomorphine displayed partial agonist activity. The dopaminergic antagonists chlorpromazine, fluphenazine, and haloperidol inhibited the DA-stimulated adenylate cyclase. There was a close correspondence between the Ka values for DA and the Ki values of the dopaminergic antagonists for particulate adenylate cyclase activity, cellular cAMP accumulation, and PTH release. These results indicate that DA-stimulated cAMP accumulation and PTH release are mediated through specific activation of a DA receptor linked to adenylate cyclase.

A familial syndrome of decrease in sensitivity to 1,25-dihydroxyvitamin D.

Typical features of hereditary vitamin D-dependent (pseudovitamin D-deficient) rickets were observed beginning at ages 20 and 5 months in a brother and sister. Both had calcium malabsorption correctable with high doses of 25-hydroxyvitamin D. During periods of hypocalcemia they both manifested secondary hyperparathyroidism with hypophosphatemia and high serum concentrations of endogenously produced 1,25-dihydroxyvitamin D. In each, normalization of serum calcium concentration and resolution of osteomalacia were obtained with continuous administration of high doses of ergocalciferol or high doses of 1,25-dihydroxycholecalciferol. Chemical features of vitamin D deficiency were corrected in the presence of high circulating concentrations of 1,25-dihydroxyvitamin D2, produced endogenously, or of 1,25-dihydroxyvitamin D3, administered by mouth. Serum concentrations of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D were normal in five first degree relatives. We conclude that in these five first degree relatives. We conclude that in these siblings, rickets and osteomalacia resulted from a hereditary decreased sensitivity to 1,25-dihydroxyvitamin D at the intestine and perhaps other vitamin D target tissues.

Immunohistochemical localization of Galphaq, PLCbeta, Galphai1-2, PKA, and the endothelin B and extracellular Ca2+-sensing receptors during early amelogenesis.

Antibodies specific to Galphaq, PLCbeta, Galphai 1-2, and PKA were immunohistochemically (IHC) localized in the pre-ameloblasts up to initial dentin matrix deposition and continued in the distal ends of the pre-secretory ameloblasts to the beginning of enamel matrix secretion. It was hypothesized that the endothelin B receptor (ETBR) and/or the extracellular Ca2+-sensing receptor (CaR) would localize in the same locations as their known downstream signal transduction pathway (STP) effectors during events related to early amelogenesis. Localization was similar for the 4 signal transduction pathway elements and the CaR. The ETBR was not localized in any of the cells of the enamel organ. These findings indicate that the CaR and its related STPs are expressed in the pre-ameloblasts and pre-secretory ameloblasts in positions where they may be able to detect increases in extracellular Ca2+ concentrations observed in the pre-dentin matrix in a previous study. These observations are consistent with the hypothesis that increased levels of free Ca2+ in the pre-dentin matrix serve as a primary signal for modification of gene expression important to amelogenesis.

Isolated C-2 osteomyelitis of hematogenous origin: case report and literature review.

A case of hematogenous Staphylococcus aureus osteomyelitis isolated to the 2nd cervical vertebra is presented. To our knowledge, this is the second case to be reported of hematogenous infection involving only the body and odontoid process of the axis. The first report was published prior to the advent of computed tomography, bone scans, and halo orthosis. The pathophysiology of blood-borne atlantoaxial infection is described, as well as methods of diagnostic evaluation and therapeutic recommendations. The use of computed tomography to define the extent of the lesion is illustrated.

Imaging of human tooth enamel using ultrasound.

This paper reports the results of a complete circumferential scan of a human tooth and its underlying dentino-enamel junction using ultrasound at frequencies in the 10-MHz range. The imagery shows clearly a two-dimensional contour of the dentinoenamel junction with a depth and lateral resolution of approximately 100 microm and 750 microm, respectively. The resulting sonograph is compared with an optical micrograph of the same tooth to verify the accuracy of the ultrasonic technique. The results are a significant step toward the biolocation of submillimeter size features within the tooth volume.

Prothrombin fragment 1-membrane interactions: a calcium-43 NMR study.

The 43Ca NMR spectra are reported for solutions of prothrombin fragment 1 in the presence and absence of phospholipid. The calcium NMR spectrum permits distinction between three thermodynamic classes of calcium-binding interactions. The calcium ion in the lipid-free solutions was labile, with maximum residence times estimated for the average protein site in the range of 0.5-1 ms. The calcium spectrum was sensitive to the protein association and the addition of phospholipid, which appears to sharpen the calcium specificity for the protein sites. The calcium NMR spectra in the presence of phospholipid are similar to those in lipid-free solutions, which suggests that the calcium ion remains labile in the lipid-protein complex.

Vocal cord injection in the treatment of acute and chronic aspiration.

The problem of maintenance of proper tracheobronchial toilet is frequently a determining factor in the morbidity and mortality of patients with vocal cord paralysis. Aspiration from an incompetent glottis can cause pneumonitis and its attendant complications. Standard management of tracheobronchial toilet in patients with vocal cord paralysis has involved direct or indirect suctioning of the trachea; however, only the symptoms and not the anatomic defect are treated by these measures. Largely due to the work of Arnold and Lewy, the technique of vocal cord injection has been advocated as a method of correcting the anatomic deficiencies in patients with vocal cord paralysis, and has been advocated in the past to prevent recurrent and chronic aspiration. This study has shown that vocal cord injection increases the ability to maintain maximum peak intraluminal air pressures following injection. It has also shown that there is an increased ability to maintain air flow by glottic closure following injection. Eleven patients were studied, each of whom has been evaluated separately in this paper. The use of vocal cord injection should be more widely used by otolaryngologists and chest disease specialists for treatment of physiologic problems as well as correcting vocal disturbances.

Target outcomes for long-term oral health care in dementia: a Delphi approach.

This study developed a list of target outcomes for long-term oral health care in persons with dementia. A three-round Delphi study was used to develop a list of target outcomes. Participants included 99 staff and 171 family members associated with the Dementia Special Care Unit in Bedford, MA. In Round 1 participants were asked to list five outcomes for long-term oral health care. Items were grouped, redundancies removed, and fed back in Round 2, when participants scored the items from 1 (least important) to 10 (most important). Round 2 responses were tabulated and the top 20 were fed back for scoring in Round 3. The top 10 target outcomes in decreasing order of importance were: patient will be free from oral pain, patient will not be at risk for aspiration, emergency dental treatment will be available when needed, prevent mouth infections, daily mouth care is as much a part of daily care as shaving or brushing hair, prevent discomfort from loose teeth or sore gums, teeth will be brushed thoroughly once a day, staff will be able to provide oral hygiene care as needed, provide dental care to prevent problems eating, and recognize oral problems early. Family and professional caregivers were remarkably consistent in their identification of the top 10 outcomes. Further work is needed to ensure broad international and interdisciplinary acceptance (including families and the long-term care residents themselves) of target outcomes for long-term oral health care in persons with dementia.

Antidepressant-induced bruxism successfully treated with gabapentin.

BACKGROUND: Symptoms consistent with bruxism are a common chief complaint in dental practice. The authors describe a case of bruxism likely induced by the antidepressant venlafaxine and successfully treated with gabapentin. CASE DESCRIPTION: A case of bruxism, anxiety, insomnia and tremor is reported in a man with bipolar disorder that developed a few days after he initiated venlafaxine therapy for depression. The patient's psychiatrist prescribed gabapentin for anxiety symptoms, and shortly thereafter the man experienced a complete resolution of the bruxism. CLINICAL IMPLICATIONS: On the basis of this case and the available literature, the authors conclude that bruxism secondary to antidepressant therapy may be common. Thus, dentists should inquire about the use of these medications in patients who have bruxism. Gabapentin may offer promise in the treatment of this condition.

Powered vs manual tooth brushing in fixed appliance patients: a short term randomized clinical trial.

Sixty-three orthodontic patients wearing upper and lower fixed appliances were randomly assigned to use either a powered toothbrush fitted with a modified orthodontic brush head (Braun Oral-B Plaque Remover 3D) or a manual toothbrush (Reach Compact Medium). A trained hygienist instructed each patient on the proper use of the allocated brush. Measurements of plaque and gingival health were made at baseline, at four weeks, and at eight weeks. Data for each group were analyzed using paired t-tests. Patients using the powered toothbrush showed a significant reduction in percentage interdental bleeding scores from baseline to four weeks (-12.7, P = .003) and this was still apparent at eight weeks (-8.6, P = .028), although there were no statistically significant changes in either plaque or gingivitis scores for this group. Those patients using a manual toothbrush showed a significant reduction in mean plaque score from baseline (four weeks = -0.18, P = < .001; eight weeks = -0.12, P = .016), but gingivitis scores were only reduced significantly at four weeks. In this group, interdental bleeding scores reduced significantly at four weeks (P = .028), but were not significantly different from baseline at eight weeks (P = .0319). When the two patient groups were compared using two sample t-tests, there were no significant differences in any of the parameters measured at any time point in the study. Over an eight-week period, there were no measurable differences between the powered toothbrush with modified orthodontic brush head and a manual toothbrush with respect to mean change in plaque, gingivitis, or interdental bleeding scores when used by patients wearing fixed appliances.

Factors affecting dentist participation in a state Medicaid program.

Provider participation is one factor affecting access to care for Medicaid recipients. There is evidence that providers are increasingly limiting their acceptance of Medicaid patients. Reasons cited for physicians and dentists not participating in Medicaid include low reimbursement rates, excessive paperwork, denial of reimbursement, and bureaucratic complexities. Telephone interviews were conducted with 92 dentists in California to determine factors affecting their decisions to participate in the California Medicaid (Medi-Cal) program. Low fees, denial of payment, and broken appointments by patients were identified as the three most important problems with the program. Non-participating dentists were more concerned about broken appointments, and complicated paperwork while less likely to believe the complexity of the program had recently decreased. Participating dentists were more concerned about the lack of services covered by Medi-Cal. The fact that participating and non-participating dentist have similar concerns about most aspects of the program may indicate that dentists who currently participate in Medi-Cal may become non-participants if problems with the program are not addressed.