BACKGROUND

DNA-based vaccines have been safe but weakly immunogenic in humans to date.

RESULTS

We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP) in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines.

CONCLUSIONS

This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate.

BACKGROUND

ClinicalTrials.gov NCT00545987.

Prostaglandin E2 (PGE2) is one of the most typical lipid mediators produced from arachidonic acid (AA) by cyclooxygenase (COX) as the rate-limiting enzyme, and acts on four kinds of receptor subtypes (EPEPEP subtype have been elucidated by studies using mice deficient in each EP subtype as well as several compounds highly selective to each EP subtype, and their findings now enable us to discuss how PGE2 initiates and exacerbates inflammation at the molecular level. Here, we review the recent advances in PGE2 receptor research by focusing on the activation of mast cells via the EPEPEP receptors. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Many mutant mice deficient in leukocyte adhesion molecules display altered hematopoiesis and neutrophilia. This study investigated whether peripheral blood neutrophil concentrations in these mice are elevated as a result of accumulation of neutrophils in the circulation or altered hematopoiesis mediated by a disrupted regulatory feedback loop. Chimeric mice were generated by transplanting various ratios of CD18(+/+) and CD18(-/-) unfractionated bone marrow cells into lethally irradiated wild-type mice, resulting in approximately 0%, 10%, 50%, 90%, or 100% CD18 null neutrophils in the blood. The presence of only 10% CD18(+/+) neutrophils was sufficient to prevent the severe neutrophilia seen in mice reconstituted with CD18(-/-) bone marrow cells. These data show that the neutrophilia in CD18(-/-) mice is not caused by enhanced neutrophil survival or the inability of neutrophils to leave the vascular compartment. In CD18(-/-), CD18(-/-)E(-/-), CD18(-/-)P(-/-), EP(-/-), and EPI(-/-) mice, levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-17 (IL-17) were elevated in proportion to the neutrophilia seen in these mice, regardless of the underlying mutation. Antibiotic treatment or the propensity to develop skin lesions did not correlate with neutrophil counts. Blocking IL-17 or G-CSF function in vivo significantly reduced neutrophil counts in severely neutrophilic mice by approximately 50% (P <.05) or 70% (P <.01), respectively. These data show that peripheral blood neutrophil numbers are regulated by a feedback loop involving G-CSF and IL-17 and that this feedback loop is disrupted when neutrophils cannot migrate into peripheral tissues.

BACKGROUND

To determine whether proton radiotherapy has clinical advantages over photon radiotherapy, we modeled the dose characteristics of both to critical normal tissue volumes using data from patients with four types of childhood brain tumors.

METHODS

Three-dimensional imaging and treatment planning data, including targeted tumor and normal tissues contours, were acquired for 40 patients, 10 each with optic pathway glioma (OPG), craniopharyngioma (CR), infratentorial ependymoma (EP), or medulloblastoma (MB). Dose-volume data were collected for the entire brain, temporal lobes, cochlea, and hypothalamus from each patient. The data were averaged and compared based on treatment modality (protons vs. photons) using dose-cognitive effects models. Outcomes were estimated over 5 years.

RESULTS

Relatively small critical normal tissue volumes such as the cochlea and hypothalamus may be spared from radiation exposure when not adjacent to the primary tumor volume. Larger normal tissue volumes such as the supratentorial brain or temporal lobes receive less of the low and intermediate doses. When applied to longitudinal models of radiation dose-cognitive effects, these differences resulted in clinically significant higher IQ scores for patients with MB and CR and academic reading scores in patients with OPG. Extreme differences between proton and photon dose distributions precluded meaningful comparison of protons and photons for patients with EP.

CONCLUSIONS

Differences in the overall dose distributions, as indicated by modeling changes in cognitive function, showed that a reduction in the lower-dose volumes or mean dose would have long-term, clinical advantages for children with MB, CR, and OPG.

Epithelial stem cells (EP-SCs) located in the bulge region of a hair follicle (HF) have the potential to give rise to hair follicle stem/progenitor cells that migrate down to regenerate HFs. Bone morphogenetic protein (BMP) signaling has been shown to regulate the HF cycle by inhibiting anagen induction. Here we show that active BMP signaling functions to prevent EP-SC activation and expansion. Dynamic expression of Noggin, a BMP antagonist, releases EP-SCs from BMP-mediated restriction, leading to EP-SC activation and initiation of the anagen phase. Experimentally induced conditional inactivation of the BMP type IA receptor (Bmpr1a) in EP-SCs leads to overproduction of HF stem/progenitor cells and the eventual formation of matricomas. This genetic manipulation of the BMP signaling pathway also reveals unexpected activation of beta-catenin, a major mediator of Wnt signaling. We propose that BMP activity controls the HF cycle by antagonizing Wnt/beta-catenin activity. This is at least partially achieved by BMP-mediated enhancement of transforming growth factor-beta-regulated epithelial cell-specific phosphatase (PTEN) function. Subsequently, PTEN, through phosphatidyl inositol 3-kinase-Akt, inhibits the activity of beta-catenin, the convergence point of the BMP and Wnt signaling pathways.

Targeting of soluble proteins to the plant vacuole is mediated by determinants that reside in the polypeptide. We identified the vacuolar targeting determinant of aleurain, a plant vacuolar thiol protease, by incorporating different sequences from proaleurain into the secreted thiol protease, proendoproteinase B (proEP-B), and vice versa. The targeting fates of the chimeric proteins were analyzed by transient expression in electroporated tobacco protoplasts. The targeting determinant SSSSFADSNPIR is positioned at the N terminus of the aleurain propeptide, and its substitution into the propeptide of EP-B caused vacuolar targeting of the resulting chimeric protein. This determinant can be divided into two smaller determinants, SSSSFADS and SNPIR, each of which is sufficient to target proEP-B chimeras to the vacuole, but with lower efficiency. These smaller determinants interact in a positive manner because the combined determinant SSSSFADSNPIR targeted proEP-B with an efficiency greater than each of the smaller determinants alone. Accordingly, the efficiency of aleurain targeting was decreased when either of the smaller determinants was disrupted by replacement with similarly positioned proEP-B sequences. Further experiments on proaleurain identified an additional determinant, VTDRAAST, adjacent to the SSSSFADSNPIR determinant that is also necessary for efficient vacuolar targeting. Our results provide evidence that efficient vacuolar targeting of this thiol protease in plant cells is mediated by the combined action of smaller contiguous determinants; two of these alone are sufficient for vacuolar targeting.

Aripiprazole (OPC 14597) is an antipsychotic drug that has high affinity for dopamine D2 and D3 receptors and the dopamine autoreceptor. It is being developed for treatment of patients with schizophrenia. The purpose of this study was to determine whether a dose response following graduated doses of aripiprazole could be quantified and correlated with its occupancy of the D2 and D3 dopamine receptors in the brain of living humans. Dopamine D2 and D3 receptor occupancy in fifteen normal male human brains was measured using positron emission tomography (PET) with [11C]raclopride. PET studies were performed before and after two weeks of administration of aripiprazole. The dopamine D2 receptor occupancy was quantified with two kinetic modeling methods without using a blood input function. Administration of aripiprazole for 14 days resulted in a dose-dependent receptor occupancy between 40 - 95% after the administration of 0.5mg, 1 mg, 2 mg, 10 mg, and 30 mg per day. These results suggest that an adequate occupancy can be obtained, and this may be useful to predict an appropriate therapeutic dose for an individual patient. Interestingly, even at striatal D2 receptor occupancy values above 90%, which occurred with the higher doses, extrapyramidal side effects (EPS) were not observed. This underlines aripiprazole's unique mechanism of action as a partial dopamine receptor agonist, which might become a novel principle in the treatment of schizophrenia.

OBJECTIVE

To determine whether AD neurofibrillary pathology influences clinical diagnostic accuracy in dementia with Lewy bodies (DLB).

BACKGROUND

Pathologic diagnosis of DLB mandates Lewy bodies but also allows for AD pathology in the form of plaques and tangles. Because clinical diagnostic accuracy of DLB remains low, the authors questioned whether the severity of AD pathology in the form of tangles might affect the clinician's ability to correctly diagnose DLB in life.

METHODS

Ninety-eight subjects with autopsy-proven DLB who had been evaluated annually at the University of California San Diego AD Research Center were identified. The clinical diagnosis used was the last diagnosis before death. Pathologic diagnosis of DLB was made according to Consensus guidelines, and Braak staging was used to assess the degree of neurofibrillary AD pathology. The clinical characteristics of subjects with DLB with low vs high Braak stages were compared and the clinical diagnostic accuracy for subjects stratified according to Braak stage was determined.

RESULTS

Only 27% of the subjects with DLB demonstrated both visual hallucinations and spontaneous extrapyramidal signs (EPS). The low Braak stage (0 to 2, n = 24) subjects had a higher frequency of visual hallucinations (65%) than did subjects with DLB with higher (3 to 6, n = 66) Braak stages (33%, p = 0.008), and showed a slightly greater but not significant degree of EPS. Although clinical diagnostic accuracy for DLB was relatively low (49%), it was higher for subjects with low (75%) compared to high (39%) Braak stages (p = 0.0039).

CONCLUSIONS

The degree of concomitant AD tangle pathology has an important influence on the clinical characteristics and, therefore, the clinical diagnostic accuracy of DLB.

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.

OBJECTIVE

Clinical features characteristic of small-cell prostate carcinoma (SCPC), "anaplastic," often emerge during the progression of prostate cancer. We sought to determine the efficacy of platinum-based chemotherapy in patients meeting at least one of seven prospectively defined "anaplastic" clinical criteria, including exclusive visceral or predominantly lytic bone metastases, bulky tumor masses, low prostate-specific antigen levels relative to tumor burden, or short response to androgen deprivation therapy.

METHODS

A 120-patient phase II trial of first-line carboplatin and docetaxel (CD) and second-line etoposide and cisplatin (EP) was designed to provide reliable clinical response estimates under a Bayesian probability model with early stopping rules in place for futility and toxicity.

RESULTS

Seventy-four of 113 (65.4%) and 24 of 71 (33.8%) were progression free after four cycles of CD and EP, respectively. Median overall survival (OS) was 16 months [95% confidence interval (CI), 13.6-19.0 months]. Of the seven "anaplastic" criteria, bulky tumor mass was significantly associated with poor outcome. Lactic acid dehydrogenase strongly predicted for OS and rapid progression. Serum carcinoembryonic antigen (CEA) concentration strongly predicted OS but not rapid progression. Neuroendocrine markers did not predict outcome or response to therapy.

CONCLUSIONS

Our findings support the hypothesis that patients with "anaplastic" prostate cancer are a recognizable subset characterized by a high response rate of short duration to platinum-containing chemotherapies, similar to SCPC. Our results suggest that CEA is useful for selecting therapy in men with castration-resistant prostate cancer and consolidative therapies to bulky high-grade tumor masses should be considered in this patient population.

An endocochlear potential (EP) of +80 mV is essential for audition. Although the regulation of K(+) concentration ([K(+)]) in various compartments of the cochlear stria vascularis seems crucial for the formation of the EP, the mechanism remains uncertain. We have used multibarreled electrodes to measure the potential, [K(+)], and input resistance in each compartment of the stria vascularis. The stria faces two fluids, perilymph and endolymph, and contains an extracelluar compartment, the intrastrial space (IS), surrounded by two epithelial layers, the marginal cell (MC) layer and that composed of intermediate and basal cells. Fluid in the IS exhibits a low [K(+)] and a positive potential, called the intrastrial potential (ISP). We found that the input resistance of the IS was high, indicating this space is electrically isolated from the neighboring extracellular fluids. This arrangement is indispensable for maintaining positive ISP. Inhibiting the K(+) transporters of the stria by anoxia, ouabain, or bumetanide caused the [K(+)] of the IS to increase and the intracellular [K(+)] of MCs to decrease, reducing both the ISP and the EP. Calculations indicate that the ISP represents the K(+) diffusion potential across the apical membranes of intermediate cells through Ba(2+)-sensitive K(+) channels. The K(+) diffusion potential across the apical membranes of MCs also contributes to the EP. Because the EP depends on two K(+) diffusion potentials and an electrical barrier in the stria vascularis, interference with any of these elements can interrupt hearing.

Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.

BACKGROUND

Plaque rupture with subsequent thrombosis is recognized as the underlying pathophysiology of most acute coronary syndromes and stroke. Thus, direct thrombus visualization may be beneficial for both diagnosis and guidance of therapy. We sought to test the feasibility of direct imaging of acute and subacute thrombosis using MRI together with a novel fibrin-binding gadolinium-labeled peptide, EP-1873, in an experimental animal model of plaque rupture and thrombosis.

RESULTS

Fifteen male New Zealand White rabbits (weight, approximately 3.5 kg) were made atherosclerotic by feeding a high-cholesterol diet after endothelial aortic injury. Plaque rupture was then induced with the use of Russell's viper venom (RVV) and histamine. Subsequently, MRI of the subrenal aorta was performed before RVV, after RVV, and after EP-1873. Histology was performed on regions suggested by MRI to contain thrombus. Nine rabbits (60%) developed plaque rupture and thrombus, including 25 thrombi visually apparent on MRI as "hot spots" after injection of EP-1873. Histological correlation confirmed all 25 thrombi (100%), with no thrombi seen in the other regions of the aorta. In the remaining 6 rabbits (control) without plaque rupture, no thrombus was observed on the MR images or on histology.

CONCLUSIONS

We demonstrate the feasibility of in vivo "molecular" MRI for the detection of acute and subacute thrombosis using a novel fibrin-binding MRI contrast agent in an animal model of atherosclerosis and acute/subacute thrombosis. Potential clinical applications include thrombus detection in acute coronary syndromes and stroke.

Titanium (Ti) surface roughness affects proliferation, differentiation, and matrix production of MG-63 osteoblast-like cells. Cytokines and growth factors produced in the milieu surrounding an implant may also be influenced by its surface, thereby modulating the healing process. This study examined the effect of surface roughness on the production of two factors known to have potent effects on bone, prostaglandin E2 (PGE2) and transforming growth factor beta 1 (TGF-beta 1). MG-63 cells were cultured on Ti disks of varying roughness. The surfaces were ranked from smoothest to roughest: electropolished (EP), pretreated with hydrofluoric acid-nitric acid (PT), fine sand-blasted, etched with HCl and H2SO4, and washed (EA), coarse sand-blasted, etched with HCl and H2SO4, and washed (CA), and Ti plasma-sprayed (TPS). Cells were cultured in 24-well polystyrene (plastic) dishes as controls and to determine when confluence was achieved. Media were collected and cell number determined 24 h postconfluence. PGE2 and TGF-beta 1 levels in the conditioned media were determined using commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. There was an inverse relationship between cell number and Ti surface roughness. Total PGE2 content in the media of cultures grown on the three roughest surfaces (FA, CA, and TPS) was significantly increased 1.5-4.0 times over that found in media of cultures grown on plastic or smooth surfaces. When PGE2 production was expressed per cell number, CA and TPS cultures exhibited six- to eightfold increases compared to cultures on plastic and smooth surfaces. There was a direct relationship between TGF-beta 1 production and surface roughness, both in terms of total TGF-beta 1 per culture and when normalized for cell number. TGF-beta 1 production on rough surfaces (CA and TPS) was three to five times higher than on plastic. These studies indicate that substrate surface roughness affects cytokine and growth factor production by MG-63 cells, suggesting that surface roughness may modulate the activity of cells interacting with an implant, and thereby affect tissue healing and implant success.

"Atypical" antipsychotics represent a new generation of antipsychotics with a significantly lower incidence of extrapyramidal side effects (EPS), as well as little or no effect on prolactin elevation. These advantages constitute a major improvement in the treatment of patients with schizophrenia. The exact mechanisms that make these drugs atypical is not clear. However, a preferential action on serotonin 5-HT2 or D4 receptors, or a more rapid dissociation from the dopamine D2 receptor, may account for atypicality. Although the atypical antipsychotics have overcome EPS, other side effects such as weight gain and impaired glucose tolerance/lipid abnormalities have come to the fore. Thus, the challenges are far from over. The current atypicals are much more effective against the psychosis of schizophrenia than against the other, more enduring aspects of this disorder, e.g. negative symptoms and cognitive dysfunction. At present, the atypicals use a "pharmacological shotgun" strategy to treat aspects of the disease in all patients. A more sophisticated and perhaps effective approach to schizophrenia may lie in independently targeting the pathophysiological mechanisms of each clinical dimension (i.e. positive, negative, cognitive, and affective) with more selective drugs that can be combined and individually titrated to the needs of each patient.

Alterations in neural activity due to pain and injury in early development may produce long-term effects on sensory processing and future responses to pain. To investigate persistent alterations in sensory perception, we performed quantitative sensory testing (QST) in extremely preterm (EP) children (n=43) recruited from the UK EPICure cohort (born less than 26 weeks gestation in 1995) and in age and sex matched term-born controls (TC; n=44). EP children had a generalized decreased sensitivity to all thermal modalities, but no difference in mechanical sensitivity at the thenar eminence. EP children who also required neonatal surgery had more marked thermal hypoalgesia, but did not differ from non-surgical EP children in the measures of neonatal brain injury or current cognitive ability. Adjacent to neonatal thoracotomy scars there was a localized decrease in both thermal and mechanical sensitivity that differed from EP children with scars relating to less invasive procedural interventions or from those without scars. No relationship was found between sensory perception thresholds and current pain experience or pain coping styles in EP or TC children. Neonatal care and surgery in EP children are associated with persistent modality-specific changes in sensory processing. Decreases in mechanical and thermal sensitivity adjacent to scars may be related to localized tissue injury, whereas generalized decreases in thermal sensitivity but not in mechanical sensitivity suggest centrally mediated alterations in the modulation of C-fibre nociceptor pathways, which may impact on responses to future pain or surgery.

BACKGROUND

Encapsulating peritoneal sclerosis (EPS) is recognized as a rare but serious complication of peritoneal dialysis (PD). The aim of this study was to determine the incidence, clinical features, and mortality rate of EPS.

METHODS

The authors requested the registration of all PD patients in facilities across Japan where more than 10 patients were treated with PD in this prospective multicenter study. During the 4-year study, the incidence of EPS was observed in the enrolled patients.

RESULTS

A total of 1,958 patients who were treated with PD in 57 facilities were followed up from April 1999 through March 2003. EPS occurred in 48 patients, corresponding to an overall incidence of 2.5%. In 33 of the 48 (68.8%) patients, EPS was found after discontinuation of PD. The incidence (and mortality rate) of EPS was 0%, 0.7% (0%), 2.1% (8.3%), 5.9% (28.6%), 5.8% (61.5%), and 17.2% (100%) in patients who had undergone PD for 3, 5, 8, 10, 15, and more than 15 years, respectively. The recovery ratio with total parenteral nutrition, corticosteroids and surgical treatment were 0%, 38.5%, and 58.3%, respectively. Eighteen patients (37.5%) died, 22 (45.8%) recovered, and the status of the other 8 (16.7%) remained unchanged.

CONCLUSIONS

The results of this prospective multicenter study showed that the incidence of EPS was 2.5% within a 4-year observation period and that two thirds of the cases were diagnosed after discontinuation of PD. Because of the current progress in diagnostic technology and therapeutic methodology, it appears that PD can be continued successfully with an acceptable, low risk for EPS for at least 8 years, whereas stricter caution is required for patients receiving PD for longer periods.

Protein kinase-B (PKB) and its target, the forkhead transcription factor like 1 (FKHRL1)/FoxO3a, have been suggested as regulators of neurotrophin-mediated cell survival in neuronal cells. We analyzed human neuroblastoma cells and found that FKHRL1 was phosphorylated, suggesting its inactivation. To study FKHRL1 function, we infected SH-EP and NB15 cells with a 4OH-tamoxifen-regulated FKHRL1(A3)ER(tm) transgene. Activation of FKHRL1 promoted cytochrome-c release and caspase-dependent apoptosis. FKHRL1 induced TRAIL and the BH3-only proteins Noxa and Bim, implicating both extrinsic and intrinsic death pathways. However, expression of dnFADD did not inhibit FKHRL1-induced cell death, whereas Bcl2 protected against apoptosis. This excluded the death-receptor pathway and suggested that cell death decision is regulated by Bcl2-rheostat. Importantly, RNAi knockdown of Noxa or Bim decreased apoptosis, indicating that Noxa and Bim cooperate to mediate FKHRL1-induced cell death. We conclude that Noxa and Bim establish a connection between FKHRL1 and mitochondria, and that both BH3-only proteins are critically involved in FKHRL1-induced apoptosis in neuroblastoma.

Ep-CAM is a homophilic, Ca2+-independent cell-cell adhesion molecule that is expressed in many human epithelial tissues. Its increased expression is closely associated with active cell proliferation. Furthermore, in epithelial cell types that in adults lack Ep-CAM (i. e. squamous epithelia), up-regulation of Ep-CAM coincides with the early stages of neoplastic change. This study has analysed the expression of Ep-CAM in liver, in the hepatocytes and cells of the biliary duct system, in relation to proliferative diseases and carcinogenesis. Adult hepatocytes are Ep-CAM negative, with only bile duct epithelium being positive in the liver tissue. However, in the 8-week embryonic liver, the majority of hepatocytes express Ep-CAM. During regeneration and repair of liver tissues associated with focal nodular hyperplasia and (biliary) cirrhosis, activation of Ep-CAM expression was observed, with high expression levels in so-called 'ductular proliferations'-regenerating stem cells. During precursor cell differentiation into mature hepatocytes, several intermediate morphological stages could be observed, all Ep-CAM positive, including cells morphologically close to mature hepatocytes. Full maturation of the precursor resulted in the disappearance of Ep-CAM expression. The results suggest that expression of Ep-CAM is a prerequisite of the proliferative phenotype during differentiation of hepatocyte precursors. In liver neoplasia, Ep-CAM was expressed in almost all cholangiocarcinomas (10/11), whereas the majority of hepatocellular carcinomas (8/10) were negative, suggesting that malignant proliferation of hepatocellular carcinoma cells is not related to expression of Ep-CAM and that hepatocellular carcinoma originates from a highly differentiated precursor. The results indicate that Ep-CAM can be used as an additional immunohistochemical marker to distinguish cholangiocarcinoma from hepatocellular carcinoma due to the differential expression of Ep-CAM in these tumours.

The rapid identification of genetic markers for multifactorial diseases from genome-wide association studies is fuelling interest in investigating the predictive ability and health care utility of genetic risk models. Various measures are available for the assessment of risk prediction models, each addressing a different aspect of performance and utility. We developed PredictABEL, a package in R that covers descriptive tables, measures and figures that are used in the analysis of risk prediction studies such as measures of model fit, predictive ability and clinical utility, and risk distributions, calibration plot and the receiver operating characteristic plot. Tables and figures are saved as separate files in a user-specified format, which include publication-quality EPS and TIFF formats. All figures are available in a ready-made layout, but they can be customized to the preferences of the user. The package has been developed for the analysis of genetic risk prediction studies, but can also be used for studies that only include non-genetic risk factors. PredictABEL is freely available at the websites of GenABEL ( http://www.genabel.org ) and CRAN ( http://cran.r-project.org/).

The electropermeabilization (EPN) of living cells allows the uptake of non-permeant molecules and can reveal their potential activity on cells without the constraints of the plasma membrane crossing. We decided to compare the cytotoxicity of some anticancer drugs on electropermeabilized (EP) and non-permeabilized (NEP) cultured DC-3F cells exposed to the drugs for a short time. After EPN, the increase in cytotoxicity varies between 1 and more than 700 times, depending on the usual cell uptake pathway of a given drug. The most relevant increase of toxicity was observed with molecules such as netropsin (200-fold) and bleomycin (700-fold) which in ordinary conditions weakly diffuse through the plasma membrane. Only a 3-5-fold increase of the cytotoxicity was observed with lipophilic drugs able to rapidly diffuse through the plasma membrane (actinomycin D, NMHE) both in the case of drug-sensitive and resistant cell strains. This increased toxicity is clearly related to a facilitated uptake because, after electropermeabilization, the effects of melphalan (a drug which enters intact cells via leucine transporters) are not modulated by the external leucine concentration. Thus, EPN enables us to reveal the intrinsic toxicity of hydrophilic molecules which have a limited access to their intracellular targets. We propose that EPN can be used as a novel screening procedure of new cytotoxic molecules which could be modified thereafter in order to facilitate their cellular uptake.

Anemia and iron deficiency during pregnancy are prevalent in developing countries, but their causes are not always known. We assessed the prevalence and severity of anemia and iron deficiency and their association with helminths, malaria and vitamin A deficiency in a community-based sample of 336 pregnant women in the plains of Nepal. Hemoglobin, erythrocyte protoporphyrin (EP) and serum ferritin were assessed in venous blood samples. Overall, 72.6% of women were anemic (hemoglobin < 110 g/L), 19.9% had moderate to severe anemia (hemoglobin < 90 g/L) and 80.6% had iron deficiency (EP>> 70 micromol/mol heme or serum ferritin < 10 microg/L). Eighty-eight percent of cases of anemia were associated with iron deficiency. More than half of the women (54.2%) had a low serum retinol concentration (<1.05 micromol/L), 74.2% were infected with hookworms and 19.8% had Plasmodium vivax malaria parasitemia. Hemoglobin, EP and serum ferritin concentrations were significantly worse and the prevalence of anemia, elevated EP and low serum ferritin was increased with increasing intensity of hookworm infection. Hookworm infection intensity was the strongest predictor of iron status, especially of depleted iron stores. Low serum retinol was most strongly associated with mild anemia, whereas P. vivax malaria and hookworm infection intensity were stronger predictors of moderate to severe anemia. These findings reinforce the need for programs to consider reducing the prevalence of hookworm, malaria infection and vitamin A deficiency where indicated, in addition to providing iron supplements to effectively control anemia.

BACKGROUND

Cardiac sarcoidosis (CS) causes substantial morbidity and sudden death. Early diagnosis and risk stratification are warranted.

METHODS

Ambulatory patients with sarcoidosis were interviewed to determine whether they experienced palpitations, syncope, or presyncope, and were evaluated with ECG, Holter monitoring, and echocardiography (transthoracic echocardiogram [TTE]). Those with symptoms or abnormal results were studied with cardiac MRI (CMRI) or positron emission tomography (PET) scanning. The diagnosis of CS was based on abnormalities detected by these imaging studies. Patients with CS were referred for risk stratification by electrophysiology study (EPS).

RESULTS

Among the 62 patients evaluated, the prevalence of CS was 39%. Patients with CS had more cardiac symptoms than those without CS (46% vs 5%, respectively; p < 0.001), and were more likely to have abnormal Holter monitoring findings (50% vs 3%, respectively; p < 0.001) and TTE findings (25% vs 5%, respectively; p = 0.02). The degree of pulmonary impairment did not predict CS. Two of the 17 patients who underwent EPS had abnormal test findings and received implantable cardioverter-defibrillators. No patients died, had ventricular arrhythmias that triggered defibrillator therapy, or had heart failure develop during almost 2 years of follow-up. This diagnostic approach was more sensitive than the established criteria for identifying CS.

CONCLUSIONS

CS is common among patients with sarcoidosis. A structured clinical assessment incorporating advanced cardiac imaging with PET scanning or CMRI is more sensitive than the established criteria for the identification of CS. Sarcoidal lesions seen on CMRI or PET scanning do not predict arrhythmias in ambulatory patients with preserved cardiac function, who appear to be at low risk for short-term mortality.

We describe the genetic and kinetic defects for a low-affinity fast channel disease of the acetylcholine receptor (AChR) that causes a myasthenic syndrome. In two unrelated patients with very small miniature end plate (EP) potentials, but with normal EP AChR density and normal EP ultrastructure, patch-clamp studies demonstrated infrequent AChR channel events, diminished channel reopenings during ACh occupancy, and resistance to desensitization by ACh. Each patient had two heteroallelic AChR epsilon subunit gene mutations: a common epsilon P121L mutation, a signal peptide mutation (epsilon G-8R) (patient 1), and a glycosylation consensus site mutation (epsilon S143L) (patient 2). AChR expression in HEK fibroblasts was normal with epsilon P121L but was markedly reduced with the other mutations. Therefore, epsilon P121L defines the clinical phenotype. Studies of the engineered epsilon P121L AChR revealed a markedly decreased rate of channel opening, little change in affinity of the resting state for ACh, but reduced affinity of the open channel and desensitized states.