Immunogenicity, including vitiligo, and feasibility of vaccination with autologous GM-CSF-transduced tumor cells in metastatic melanoma patients.

PURPOSE: To determine the feasibility, toxicity, and immunologic effects of vaccination with autologous tumor cells retrovirally transduced with the GM-CSF gene, we performed a phase I/II vaccination study in stage IV metastatic melanoma patients. PATIENTS AND METHODS: Sixty-four patients were randomly assigned to receive three vaccinations of high-dose or low-dose tumor cells at 3-week intervals. Tumor cell vaccine preparation succeeded for 56 patients (88%), but because of progressive disease, the well-tolerated vaccination was completed in only 28 patients. We analyzed the priming of T cells against melanoma antigens, MART-1, tyrosinase, gp100, MAGE-A1, and MAGE-A3 using human leukocyte antigen/peptide tetramers and functional assays. RESULTS: The high-dose vaccination induced the infiltration of T cells into the tumor tissue. Three of 14 patients receiving the high-dose vaccine showed an increase in MART-1- or gp100-specific T cells in the peripheral blood during vaccination. Six patients experienced disease-free survival for more than 5 years, and two of these patients developed vitiligo at multiple sites after vaccination. MART-1- and gp100-specific T cells were found infiltrating in vitiligo skin. Upon vaccination, the T cells acquired an effector phenotype and produced interferon-gamma on specific antigenic stimulation. CONCLUSION: We conclude that vaccination with GM-CSF-transduced autologous tumor cells has limited toxicity and can enhance T-cell activation against melanocyte differentiation antigens, which can lead to vitiligo. Whether the induction of autoimmune vitiligo may prolong disease-free survival of metastatic melanoma patients who are surgically rendered as having no evidence of disease before vaccination is worthy of further investigation.     

Importance of studying the contributions of early adverse experience to neurobiological findings in depression.

Almost four decades of intensive research have sought to elucidate the neurobiological bases of depression. Epidemiological studies have revealed that both genetic and environmental factors contribute to the risk for depression. Adverse early-life experiences influence neurobiological systems within genetic limits, leading to the neurobiological and behavioral manifestations of depression. We summarize the burgeoning evidence concerning a pre-eminent role of early adverse experience in the pathogenesis of depression. The available data suggest that (1) early adverse experience contributes to the pathophysiology of depression, (2) there are neurobiologically different subtypes of depression depending on the presence or absence of early adverse experience, likely having confounded previous research on the neurobiology of depression, and (3) early adverse experience likely influences treatment response in depression. Classification of depression based on developmental and neurobiological features will likely considerably improve future research in the field of depression, and might lead to optimized treatment strategies that directly target different neurobiological pathways to depression.     

Preteen children and illegal drugs

In this paper we report the results of research on the nature and extent of legal and illegal drug use among preteens and those factors associated with illegal drug use at this young age. The paper is based upon a survey of 2318 ten to twelve year olds in Glasgow and Newcastle. Overall around 30% of children reported having been exposed to illegal drugs and 3.9% had started to use illegal drugs. There was a significant difference in the level of illegal drug use between our two cities. In most cases the illegal drug use identified on the part of preteens related to cannabis although in a small number of instances children were using heroin, cocaine and LSD. On the basis of this research we estimate that around 60 children in the ten to twelve age range will have used heroin in Glasgow and around 34 pupils will have used the drug in Newcastle. Preteen drug use was significantly associated with frequent smoking and alcohol consumption, with preteens' involvement in a range of problem behaviours, and with family difficulties including the presence of someone else within the family using illegal drugs. The paper concludes by noting some of the challenges that are likely to be faced by services seeking to support children who are using illegal drugs by their preteens.     

Pharmacological Prevention of Reperfusion Injury in Acute Myocardial Infarction

The concept of reperfusion injury, although first recognized from animal studies, is now recognized as a clinical phenomenon that may result in microvascular damage, no-reflow phenomenon, myocardial stunning, myocardial hibernation and ischemic preconditioning. The final consequence of this event is left ventricular (LV) systolic dysfunction leading to increased morbidity and mortality. The typical clinical case of reperfusion injury occurs in acute myocardial infarction (MI) with ST segment elevation in which an occlusion of a major epicardial coronary artery is followed by recanalization of the artery. This may occur either spontaneously or by means of thrombolysis and/or by primary percutaneous coronary intervention (PCI) with efficient platelet inhibition by aspirin (acetylsalicylic acid), clopidogrel and glycoprotein IIb/IIIa inhibitors. Although the pathophysiology of reperfusion injury is complex, the major role that neutrophils play in this process is well known. Neutrophils generate free radicals, degranulation products, arachidonic acid metabolites and platelet-activating factors that interact with endothelial cells, inducing endothelial injury and neutralization of nitrous oxide vasodilator capacity. Adenosine, through its multi-targeted pharmacological actions, is able to inhibit some of the above-mentioned detrimental effects. The net protective of adenosine in in vivo models of reperfusion injury is the reduction of the infarct size, the improvement of the regional myocardial blood flow and of the regional function of the ischemic area. Additionally, adenosine preserves the post-ischemic coronary flow reserve, coronary blood flow and the post-ischemic regional contractility. In small-scale studies in patients with acute MI, treatment with adenosine has been associated with smaller infarcts, less no-reflow phenomenon and improved LV function. During elective PCI adenosine reduced ST segment shifts, lactate production and ischemic symptoms. During the last years, three relatively large placebo-controlled clinical trials have been conducted: Acute Myocardial Infarction Study of Adenosine Trial (AMISTAD) I and II and Attenuation by Adenosine of Cardiac Complications (ATTACC). In the AMISTAD trials, the final infarct size was reduced and the LV systolic function was improved by adenosine treatment, mainly in patients with anterior MI localization. However, morbidity and mortality were not affected. In the ATTACC study, the LV systolic function was not affected by adenosine, however, trends towards improved survival were observed in patients with anterior MI localization. The possibility of obtaining a Thrombolysis in Myocardial Infarction (TIMI) grade 3 flow in the infarct-related artery in up to 95% of patients with acute MI (increasing the occurrence of reperfusion injury) has turned back the interest towards the protection of myocardial cells from the impending ischemic and reperfusion injury in which adenosine alone or together with other cardio-protective agents may exert important clinical effects.     

The influence of humidity on the aerosolisation of micronised and SEDS produced salbutamol sulphate.

The influence of storage humidity on the aerosolisation efficiency of micronised and supercritical fluid salbutamol sulphate formulations (containing a lactose carrier) were investigated using the twin stage impinger (TSI). Storage humidity had a significant effect on the aerosolisation efficiency of both micronised and solution enhanced dispersion by supercritical fluids (SEDS) salbutamol sulphate (ANOVA P <0.05), suggesting capillary interactions to be an important factor when considering formulation performance. Furthermore, significant differences between the aerosolisation performance of micronised and SEDS salbutamol sulphate were observed at elevated humidities (>63% RH) (Fishers pairwise P <0.05). It is suggested that such variations may be due to differences in physical stability of the micronised and SEDS produced material. Dynamic vapor sorption, and atomic force microscopy (AFM) phase imaging suggested the micronised material to contain amorphous content that was most likely present on the micronised particulate surfaces. Thus, at high humidity, surface amorphous regions may have the ability to re-crystallize and effectively 'fuse' to the lactose carrier surface. This would potentially reduce the ability for the micronised material to be aerosolized and thus result in a greater decrease in FPF when compared to SEDS produced material at equivalent RH.     

Influence of the punch diameter and curvature on the yield pressure of MCC-compacts during Heckel analysis.

The volume reduction behaviour of powders has been quantified by means of the 'in-die' yield pressure (YP) using Heckel analysis. However, because different YPs are reported for the same material, the experimental conditions influencing this material-constant were investigated. Silicified microcrystalline cellulose was compressed into flat-faced and convex tablets using a compaction simulator instrumented with load and displacement transducers. During compression, upper and lower punch force and displacement data were recorded and corrected for punch deformation. A symmetrical triangle wave compression profile was used and the instantaneous punch velocity was kept constant (5mm/s). Individual tablet height and weight were used for Heckel analysis. The influence of the 'effective compression pressure' (P(EFF)) (ranging from 10 to 350 MPa), punch diameter (PD) (4, 9.5 and 12 mm) and filling depth (FD) (4.5, 7.5 and 10.5mm) on YP was statistically evaluated using Response Surface Modelling software. A quadratic surface response equation, describing the relationship between P(EFF), PD, FD and YP, was proposed for concave (Adj R(2): 0.8424; S.D.: 14.60 MPa) and flat-faced (Adj R(2): 0.8409; S.D.: 4.49 MPa) punches. YP and tensile strength were mainly determined by P(EFF), irrespective of punch curvature. FD and PD had only a minor influence on the YP, although more pronounced for the concave punches. The method used resulted in reproducible P(EFF) and tensile strength values and the flat-faced tablets showed less weight variation. Flat-faced punches are preferred over punches with a concave surface when investigating the volume reduction behaviour of a powder by means of Heckel analysis and the experimental parameters should be reported.     

Confirmation of brevetoxin metabolism in cockle, Austrovenus stutchburyi, and greenshell mussel, Perna canaliculus, associated with New Zealand neurotoxic shellfish poisoning, by controlled exposure to Karenia brevis culture.

We examined metabolism of PbTxs in New Zealand cockle, Austrovenus (A.) stutchburyi, and greenshell mussel, Perna (P.) canaliculus, by means of liquid chromatography coupled with tandem mass spectrometry. PbTx-2, PbTx-3 and BTX-B5 were detected in Karenia (K.) brevis culture medium in the ratio of ca. 50:2:5. The amounts of PbTx-3 and BTX-B5 were greatly increased in both seawater and shellfish exposed to K. brevis cultures or supernatant prepared by disruption of K. brevis under appropriate condition, while those of PbTx-2 were decreased. Some PbTx-2 was present in P. canaliculus, but not in A. stutchburyi. Low levels of BTX-B1 were detected in A. stutchburyi, but not P. canaliculus. Levels of PbTx-3 and BTX-B5 were highest immediately after exposure and then declined rapidly in both shellfish. BTX-B1 increased in concentration after exposure, and was then gradually eliminated from A. stutchburyi. Three successive exposures of A. stutchburyi to K. brevis cultures resulted in similar initial levels of PbTx-3 and BTX-B5, while BTX-B1 accumulated after each dose. In P. canaliculus, initial levels of PbTx-3 were similar, while PbTx-2 and BTX-B5 accumulated after each dose. PbTx-3 and BTX-B5 are proposed to be suitable markers for monitoring shellfish toxicity after a red tide event.     

Phytochemicals inhibit catechol-O-methyltransferase activity in cytosolic fractions from healthy human mammary tissues: implications for catechol estrogen-induced DNA damage.

Phytochemicals are natural dietary constituents of fruits and vegetables. Some of these phytochemicals are known to affect estrogen-metabolizing enzymes. In breast tissue, estradiol can be metabolized to the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE(2) and 4-OHE(2)). Catechol estrogens are suspected carcinogens potentially involved in the etiology of breast cancer. Catechol-O-methyltransferase (COMT) converts the catechol estrogens to their inactive methoxy derivatives (2-MeOE(2) and 4-MeOE(2)). In this study we investigated the effects of several phytochemicals on COMT activity in cytosolic fractions of seven healthy human mammary tissues from reduction mammoplasty. Large interindividual variations were observed in the constitutive levels of COMT activity. However, in all cytosol samples the catalytic efficiency of COMT was greater for 2-MeOE(2) formation than for 4-MeOE(2) formation. The known COMT inhibitor Ro 41-0960 and several phytochemicals with a catechol structure (quercetin, catechin, and (-)-epicatechin) concentration-dependently inhibited COMT activity, while phytochemicals without a catechol structure (genistein, chrysin, and flavone) showed no effect up to 30 microM. Distinct interindividual variations were observed in sensitivity toward COMT inhibition among the various tissue samples, as was shown by the range in IC(50) values for Ro 41-0960 (5-42 nM). The toxicological relevance of COMT inhibition and the effect of reduced inactivation of catechol estrogens was studied by determining the amount of catechol estrogen-induced DNA damage in MCF-7 cells using the comet assay. Catechol estrogens alone caused no increase of DNA damage compared with control treated cells. However, both Ro 41-0960 and quercetin caused a decrease of methoxy estradiol formation and an increase of catechol estrogen-induced DNA damage in MCF-7 cells. This suggests that phytochemicals with a catechol structure have the potential to reduce COMT activity in mammary tissues and may consequently reduce the inactivation of potentially mutagenic estradiol metabolites and increase the chance of DNA damage.     

Two major grapefruit juice components differ in intestinal CYP3A4 inhibition kinetic and binding properties.

Bergamottin (BG) and 6',7'-dihydroxybergamottin (DHB) are the most abundant furanocoumarins present in grapefruit juice and have been proposed as major intestinal CYP3A4 inhibitors contributing to grapefruit juice-drug interactions. The relative contribution of BG versus DHB to the interaction potential is unclear, in part due to inconsistencies in the literature regarding inhibitory potency. To resolve these inconsistencies, the inhibitory kinetics of each furanocoumarin toward CYP3A4 catalytic activity were systematically characterized using representative probes from two distinct CYP3A4 substrate subgroups (testosterone and midazolam). With human intestinal microsomes, DHB was a substrate-independent reversible (Ki, approximately 0.8 microM) and mechanism-based (KI, approximately 3 microM; kinact, 0.3-0.4 min(-1)) inhibitor of CYP3A4. In contrast, BG was a substrate-dependent reversible inhibitor, with a Ki (13 microM) using midazolam that was 8-fold greater than that using testosterone, but a substrate-independent mechanism-based inhibitor (KI, approximately 25 microM; kinact, approximately 0.35 min(-1)). Similar trends resulted with cDNA-expressed CYP3A4, only the KI values for BG were approximately 10-fold lower than with microsomes. This seemed to reflect a much greater degree of microsomal protein binding by BG compared with DHB. Differential inhibition kinetics and binding properties between BG and DHB could account in part for the apparent in vitro inconsistencies in the literature. Results also emphasize the importance of appropriate substrate selection when designing inhibition studies involving dietary constituents.