In vivo passage of a simian-human immunodeficiency virus (SHIV-89.6) generated a virus, SHIV-89.6P, that exhibited increased resistance to some neutralizing antibodies (G. B. Karlsson et al., J. Exp. Med. 188:1159-1171, 1998). Here we examine the range of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies to which the passaged virus became resistant and identify envelope glycoprotein determinants of antibody resistance. Compared with the envelope glycoproteins derived from the parental SHIV-89.6, the envelope glycoproteins of the passaged virus were resistant to antibodies directed against the gp120 V3 variable loop and the CD4 binding site. By contrast, both viral envelope glycoproteins were equally sensitive to neutralization by two antibodies, 2G12 and 2F5, that recognize poorly immunogenic structures on gp120 and gp41, respectively. Changes in the V2 and V3 variable loops of gp120 were necessary and sufficient for full resistance to the IgG1b12 antibody, which is directed against the CD4 binding site. Changes in the V3 loop specified complete resistance to a V3 loop-directed antibody, while changes in the V1/V2 loops conferred partial resistance to this antibody. The epitopes of the neutralizing antibodies were not disrupted by the resistance-associated changes. These results indicate that in vivo selection occurs for HIV-1 envelope glycoproteins with variable loop conformations that restrict the access of antibodies to immunogenic neutralization epitopes.

After injury, regrowth of axons in mammalian adult central nervous system is highly limited. However, in monkeys subjected to unilateral cervical lesion (C7-C8 level), neutralization of an important neurite outgrowth inhibitor, Nogo-A, stimulated axonal sprouting caudal to the lesion, accompanied by enhanced functional recovery of manual dexterity, compared with lesioned monkeys treated with a control antibody (Freund et al. [2006] Nat. Med. 12:790-792). The present study aimed at comparing the same two groups of monkeys for axonal sprouting rostral to the cervical lesion. The corticospinal tract was labeled by injecting the anterograde tracer biotinylated dextran amine into the contralesional motor cortex. The corticospinal axons were interrupted at the level of the lesion, accompanied by retrograde axonal degeneration (axon dieback), reflected by the presence of terminal retraction bulbs. The number of terminal retraction bulbs was lower in anti-Nogo-A antibody treated monkeys, and, when present, they were found closer to the lesion than in control-antibody treated monkeys. Compared with control antibody treated monkeys, the anti-Nogo-A antibody treated monkeys exhibited an increased cumulated axon arbor length and a higher number of axon arbors going in the medial direction from the white to the gray matter. Higher in the cervical cord (at C5 level), the anti-Nogo-A treatment enhanced the number of corticospinal fibers crossing the midline, suggesting axonal sprouting. Thus, the anti-Nogo-A antibody treatment enhanced axonal sprouting rostral to the cervical lesion; some of these fibers grew around the lesion and into the caudal spinal segments. These processes paralleled the observed improved functional recovery.

We investigate the persistence of a mosquito-borne disease (malaria) in a system where mosquitoes and hosts are grouped in patches containing any number of individuals. A mosquito from any one of vector patches can bite, and take blood meals, in any one of m host patches. We confirm our earlier result (C. Dye and G. Hasibeder, 1986, Trans. R. Soc. Trop. Med. Hyg. 80, 69-77) that nonhomogeneous host selection by mosquitoes leads to basic reproductive rates (which measure the persistence of infection in the system) greater than or equal to those obtained under uniform host selection. We find, in addition, that strong associations between particular groups of mosquitoes and hosts lead to still higher basic reproductive rates. Exacting fieldwork would be required to find out how much higher.

For more than a decade, we have known that the human brain harbors progenitor cells capable of becoming mature neurons in the adult human brain. Since the original landmark article by Eriksson et al. in 1998 (Nat Med 4:1313-1317), there have been many studies investigating the effect that depression, epilepsy, Alzheimer's disease, Huntington's disease, and Parkinson's disease have on the germinal zones in the adult human brain. Of particular interest is the demonstration that there are far fewer progenitor cells in the hippocampal subgranular zone (SGZ) compared with the subventricular zone (SVZ) in the human brain. Furthermore, the quantity of progenitor cell proliferation in human neurodegenerative diseases differs from that of animal models of neurodegenerative diseases; there is minimal progenitor proliferation in the SGZ and extensive proliferation in the SVZ in the human. In this review, we will present the data from a range of human and rodent studies from which we can compare the amount of proliferation of cells in the SVZ and SGZ in different neurodegenerative diseases.

OBJECTIVE

Polyphenols are plant secondary metabolites with a large variability in their chemical structure and dietary occurrence that have been associated with some protective effects against several chronic diseases. To date, limited data exist on intake of polyphenols in populations. The current cross-sectional analysis aimed at estimating dietary intakes of all currently known individual polyphenols and total intake per class and subclass, and to identify their main food sources in the European Prospective Investigation into Cancer and Nutrition cohort.

METHODS

Dietary data at baseline were collected using a standardized 24-h dietary recall software administered to 36,037 adult subjects. Dietary data were linked with Phenol-Explorer, a database with data on 502 individual polyphenols in 452 foods and data on polyphenol losses due to cooking and food processing.

RESULTS

Mean total polyphenol intake was the highest in Aarhus-Denmark (1786 mg/day in men and 1626 mg/day in women) and the lowest in Greece (744 mg/day in men and 584 mg/day in women). When dividing the subjects into three regions, the highest intake of total polyphenols was observed in the UK health-conscious group, followed by non-Mediterranean (non-MED) and MED countries. The main polyphenol contributors were phenolic acids (52.5-56.9 %), except in men from MED countries and in the UK health-conscious group where they were flavonoids (49.1-61.7 %). Coffee, tea, and fruits were the most important food sources of total polyphenols. A total of 437 different individual polyphenols were consumed, including 94 consumed at a level >1 mg/day. The most abundant ones were the caffeoylquinic acids and the proanthocyanidin oligomers and polymers.

CONCLUSIONS

This study describes the large number of dietary individual polyphenols consumed and the high variability of their intakes between European populations, particularly between MED and non-MED countries.

The performance of the site-features docking algorithm LibDock has been evaluated across eight GlaxoSmithKline targets as a follow-up to a broad validation study of docking and scoring software (Warren, G. L.; Andrews, W. C.; Capelli, A.; Clarke, B.; Lalonde, J.; Lambert, M. H.; Lindvall, M.; Nevins, N.; Semus, S. F.; Senger, S.; Tedesco, G.; Walls, I. D.; Woolven, J. M.; Peishoff, C. E.; Head, M. S. J. Med. Chem. 2006, 49, 5912-5931). Docking experiments were performed to assess both the accuracy in reproducing the binding mode of the ligand and the retrieval of active compounds in a virtual screening protocol using both the DJD (Diller, D. J.; Merz, K. M., Jr. Proteins 2001, 43, 113-124) and LigScore2 (Krammer, A. K.; Kirchoff, P. D.; Jiang, X.; Venkatachalam, C. M.; Waldman, M. J. Mol. Graphics Modell. 2005, 23, 395-407) scoring functions. This study was conducted using DJD scoring, and poses were rescored using all available scoring functions in the Accelrys LigandFit module, including LigScore2. For six out of eight targets at least 30% of the ligands were docked within a root-mean-square difference (RMSD) of 2.0 A for the crystallographic poses when the LigScore2 scoring function was used. LibDock retrieved at least 20% of active compounds in the top 10% of screened ligands for four of the eight targets in the virtual screening protocol. In both studies the LigScore2 scoring function enhanced the retrieval of crystallographic poses or active compounds in comparison with the results obtained using the DJD scoring function. The results for LibDock accuracy and ligand retrieval in virtual screening are compared to 10 other docking and scoring programs. These studies demonstrate the utility of the LigScore2 scoring function and that LibDock as a feature directed docking method performs as well as docking programs that use genetic/growing and Monte Carlo driven algorithms.

These joint practice guidelines, or procedure standards, were developed collaboratively by the European Association of Nuclear Medicine (EANM), the Society of Nuclear Medicine and Molecular Imaging (SNMMI), the European Association of Neurooncology (EANO), and the working group for Response Assessment in Neurooncology with PET (PET-RANO). Brain PET imaging is being increasingly used to supplement MRI in the clinical management of glioma. The aim of these standards/guidelines is to assist nuclear medicine practitioners in recommending, performing, interpreting and reporting the results of brain PET imaging in patients with glioma to achieve a high-quality imaging standard for PET using FDG and the radiolabelled amino acids MET, FET and FDOPA. This will help promote the appropriate use of PET imaging and contribute to evidence-based medicine that may improve the diagnostic impact of this technique in neurooncological practice. The present document replaces a former version of the guidelines published in 2006 (Vander Borght et al. Eur J Nucl Med Mol Imaging. 33:1374-80, 2006), and supplements a recent evidence-based recommendation by the PET-RANO working group and EANO on the clinical use of PET imaging in patients with glioma (Albert et al. Neuro Oncol. 18:1199-208, 2016). The information provided should be taken in the context of local conditions and regulations.

The human CRSP-<em>Med</em> coactivator complex is targeted by a diverse array of sequence-specific regulatory proteins. Using EM and single-particle reconstruction techniques, we recently completed a structural analysis of CRSP-<em>Med</em> bound to VP16 and SREBP-1a. Notably, these activators induced distinct conformational states upon binding the coactivator. Ostensibly, these different conformational states result from VP16 and SREBP-1a targeting distinct subunits in the CRSP-<em>Med</em> complex. To test this, we conducted a structural analysis of CRSP-<em>Med</em> bound to either thyroid hormone receptor (TR) or vitamin D receptor (VDR), both of which interact with the same subunit (<em>Med</em>220) of CRSP-<em>Med</em>. Structural comparison of TR- and VDR-bound complexes (at a resolution of 29 A) indeed reveals a shared conformational feature that is distinct from other known CRSP- <em>Med</em> structures. Importantly, this nuclear receptor-induced structural shift seems largely dependent on the movement of <em>Med</em>220 within the complex.

A new parallel imaging technique was implemented which can result in reduced image acquisition times in MRI. MR data is acquired in parallel using an array of receiver coils and then reconstructed simultaneously with multiple processors. The method requires the initial estimation of the 2D sensitivity profile of each coil used in the receiver array. These sensitivity profiles are then used to partially encode the images of interest. A fraction of the total number of k-space lines is consequently acquired and used in a parallel reconstruction scheme, allowing for a substantial reduction in scanning and display times. This technique is in the family of parallel acquisition schemes such as simultaneous acquisition of spatial harmonics (SMASH) and sensitivity encoding (SENSE). It extends the use of the SMASH method to allow the placement of the receiver coil array around the object of interest, enabling imaging of any plane within the volume of interest. In addition, this technique permits the arbitrary choice of the set of k-space lines used in the reconstruction and lends itself to parallel reconstruction, hence allowing for real-time rendering. Simulated results with a 16-fold increase in temporal resolution are shown, as are experimental results with a 4-fold increase in temporal resolution. Magn Reson Med 44:301-308, 2000.

Ultrasound-based mechanical strain imaging systems utilize signals from conventional diagnostic ultrasound systems to image tissue elasticity contrast that provides new diagnostically valuable information. Previous works (Hall et al 2003 Ultrasound Med. Biol. 29 427, Zhu and Hall 2002 Ultrason. Imaging 24 161) demonstrated that uniaxial deformation with minimal elevation motion is preferred for breast strain imaging and real-time strain image feedback to operators is important to accomplish this goal. The work reported here enhances the real-time speckle tracking algorithm with two significant modifications. One fundamental change is that the proposed algorithm is a column-based algorithm (a column is defined by a line of data parallel to the ultrasound beam direction, i.e. an A-line), as opposed to a row-based algorithm (a row is defined by a line of data perpendicular to the ultrasound beam direction). Then, displacement estimates from its adjacent columns provide good guidance for motion tracking in a significantly reduced search region to reduce computational cost. Consequently, the process of displacement estimation can be naturally split into at least two separated tasks, computed in parallel, propagating outward from the center of the region of interest (ROI). The proposed algorithm has been implemented and optimized in a Windows system as a stand-alone ANSI C++ program. Results of preliminary tests, using numerical and tissue-mimicking phantoms, and in vivo tissue data, suggest that high contrast strain images can be consistently obtained with frame rates (10 frames s(-1)) that exceed our previous methods.

BACKGROUND

An increase in the incidence of hepatocellular carcinoma (HCC) in Japan since the 1980s suggests an imminent outbreak in other countries where viral spread occurred more recently. Interferon therapy for chronic hepatitis C, in general, has been shown to prevent HCC.

OBJECTIVE

To determine the scale of benefit in individual patients.

METHODS

Histologically proven chronic hepatitis C patients in the Inhibition of Hepatocarcinogenesis by Interferon Therapy (IHIT) cohort (Ann Intern Med 1999;131:174), as updated in March 2003.

METHODS

The lifetime risk for HCC was calculated based on HCC incidence rates, stratified by sex, age, fibrosis stage, and outcome of interferon therapy. The gain in HCC free survival was defined as the difference between expected HCC free survival with sustained virological response and that without.

RESULTS

The gain in HCC free survival was greater when a patient was younger and fibrosis was more advanced. For example, a 30 year old male with F3 fibrosis gained 12.4 years by attaining sustained response while a patient with F1 fibrosis older than 60 years gained less than one year. For a treatment protocol with a given sustained response rate, prior estimation of the gain can be obtained by multiplying the calculated HCC free survival for responders by the response rate.

CONCLUSIONS

The gain in HCC free survival may serve as an indicator of the benefit of interferon therapy in terms of HCC prevention and be useful in the consideration of indication and selection of treatment protocol for individual patients.

The regulator of G protein signaling (RGS) molecules are a class of proteins that modulate the signaling activity of G-protein coupled receptors. Regulator of G protein signaling 4 (RGS4) is of particular interest in schizophrenia since it is associated with the dopamine (DA) receptor, its expression is altered in affected CNS tissue, and polymorphisms in the RGS4 gene are being examined as risk factors for the disease (Morris et al.2004, Am J Med Genet B Neuropsychiatr Genet 125:50-53; Prasad et al.2005, Mol Psychiatry 10:213-219; Williams et al.2004, Biol Psychiatry 55:192-195). To further test for the involvement of RGS4 expression in schizophrenia, we examined a selection of different cortical and subcortical regions in human brain for alterations in RGS4 mRNA and protein expression. To evaluate the effect of antipsychotic medication on RGS4 expression levels, we compared a subset of treated and untreated cases that were off antipsychotic medication for at least 3 months prior to death. We report a significant decrease in RGS4 mRNA levels in the cingulate gyrus, superior frontal gyrus, and the insular cortex of all schizophrenia cases when compared with controls. A decrease in RGS4 mRNA was also observed in the caudate, but only in the medicated schizophrenia cases. Measurement of protein levels using Western blot demonstrated that RGS4 protein is decreased in the frontal cortex of schizophrenia cases.

This paper describes the methodologies used to develop a prediction model to assist health workers in developing countries in facing one of the most difficult health problems in all parts of the world: the presentation of an acutely ill young infant. Statistical approaches for developing the clinical prediction model faced at least two major difficulties. First, the number of predictor variables, especially clinical signs and symptoms, is very large, necessitating the use of data reduction techniques that are blinded to the outcome. Second, there is no uniquely accepted continuous outcome measure or final binary diagnostic criterion. For example, the diagnosis of neonatal sepsis is ill-defined. Clinical decision makers must identify infants likely to have positive cultures as well as to grade the severity of illness. In the WHO/ARI Young Infant Multicentre Study we have found an ordinal outcome scale made up of a mixture of laboratory and diagnostic markers to have several clinical advantages as well as to increase the power of tests for risk factors. Such a mixed ordinal scale does present statistical challenges because it may violate constant slope assumptions of ordinal regression models. In this paper we develop and validate an ordinal predictive model after choosing a data reduction technique. We show how ordinality of the outcome is checked against each predictor. We describe new but simple techniques for graphically examining residuals from ordinal logistic models to detect problems with variable transformations as well as to detect non-proportional odds and other lack of fit. We examine an alternative type of ordinal logistic model, the continuation ratio model, to determine if it provides a better fit. We find that it does not but that this model is easily modified to allow the regression coefficients to vary with cut-offs of the response variable. Complex terms in this extended model are penalized to allow only as much complexity as the data will support. We approximate the extended continuation ratio model with a model with fewer terms to allow us to draw a nomogram for obtaining various predictions. The model is validated for calibration and discrimination using the bootstrap. We apply much of the modelling strategy described in Harrell, Lee and Mark (Statist. Med. 15, 361-387 (1998)) for survival analysis, adapting it to ordinal logistic regression and further emphasizing penalized maximum likelihood estimation and data reduction.

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.

The aim of this study was to investigate whether heterozygous reeler mice (+/rl) could be used as a genetic mouse model of schizophrenia, as previously suggested [J. Med. Chem. 44 (2001) 477]. The behavioural phenotype of male and female +/rl mice (young adult: 50-70 days old, and fully adult: >75 days old) was compared to their wild type (+/+) littermates. A complex behavioural test battery was employed: Irwin test, rotarod, spontaneous locomotor activity, social behaviour, light-dark transition test, startle response and prepulse inhibition, and hot-plate test. Overall, +/rl mice did not differ from their +/+ littermates at either age, although fully adult +/rl male mice spent more time engaged in social investigation. Some of the behavioural measures investigated were influenced by gender. Young female mice were more active in the light/dark transition test than males, while males were more aggressive than females during social interaction. In addition, performance on the rotarod was shown to deteriorate with age. Our data are in agreement with previous findings [Soc. Neurosci. Abst. 27 (2001) 238; J. Psychopharmacol. 17 (2003) A43], but contrary to those of Costa et al. [Curr. Opin. Pharmacol. 2 (2002) 56], although mice used in the present and previous studies were derived from the same genetic stock at Jackson Laboratories, USA. The present study clearly shows that, compared to its +/+ littermates, the +/rl mouse (young/fully adult) exhibits normal behaviour in a wide range of behavioural measures and suggest that these mice may not be suitable for use as a genetic animal model of schizophrenia.

OBJECTIVE

Although intensity modulated radiotherapy (IMRT) is a step forward in comparison to conventional, static beam delivery, quality assurance is more complex and labour intensive, demanding detailed two-dimensional dosimetric verification. Regardless of the technique used for measuring the dose distribution, what is essential to the implementation of routine verification of IMRT fields is the efficient and accurate comparison of the measured versus desired dose distribution. In order to achieve a fast, yet accurate quantitative measure of the correspondence between measured and calculated dose, the theoretical concept of the gamma evaluation method presented by Low et al. (Med. Phys., 25 (1998) 656) was converted into a calculation algorithm, taking into account practical considerations related to the discrete nature of the data.

METHODS

A filter cascade of multiple levels was designed to obtain fast and accurate comparison of the two dose distributions under evaluation. The actual comparison consists of classification into accepted or rejected datapoints with respect to user-defined acceptance criteria (dose difference and distance to agreement). The presented algorithm was tested on dosimetric images calculated and/or acquired by means of a liquid filled portal imaging device during the course of intensity modulated treatments of prostate cancer, including pre-treatment verification as well as verification during treatment. To assess its ability to intercept possible errors in dose delivery, clinically relevant errors were deliberately introduced into the dose distributions.

RESULTS

The developed gamma filter method proves successful in the efficient comparison of calculated versus measured IMRT dose distribution. Secondly, intercomparison of dosimetric images acquired during different treatment sessions illustrate its potential to highlight variations in the dosimetric images. The simulated errors were unmistakably intercepted.

CONCLUSIONS

The readily obtained gamma evaluation images are an easy tool for quality control of IMRT fields. To reduce the artefacts related to the discrete nature and limited resolution of the data, a fast and accurate filter cascade was developed, offering the possibility to use the gamma method for day to day evaluation of patient dosimetric portal images with or without comparison to a predicted portal dose distribution.

We analyzed a recently reported (K. Seno, T. Okuno, K. Nishi, Y. Murakami, F. Watanabe, T. Matsuur, M. Wada, Y. Fujii, M. Yamada, T. Ogawa, T. Okada, H. Hashizume, M. Kii, S.-H. Hara, S. Hagishita, S. Nakamoto, J. Med. Chem. 43 (2000)) pyrrolidine-based inhibitor, pyrrolidine-1, against the human group IV cytosolic phospholipase A(2) alpha-isoform (cPLA(2)alpha). Pyrrolidine-1 inhibits cPLA(2)alpha by 50% when present at approx. 0.002 mole fraction in the interface in a number of in vitro assays. It is much less potent on the cPLA(2)gamma isoform, calcium-independent group VI PLA(2) and groups IIA, X, and V secreted PLA(2)s. Pyrrolidine-1 blocked all of the arachidonic acid released in Ca(2+) ionophore-stimulated CHO cells stably transfected with cPLA(2)alpha, in zymosan- and okadaic acid-stimulated mouse peritoneal macrophages, and in ATP- and Ca(2+) ionophore-stimulated MDCK cells.

Wild-type Escherichia coli was not motile when grown in tryptone broth under the following adverse conditions: the presence of high temperature [J. Adler and B. Templeton, J. Gen. Microbiol. 46:175-184, 1967; R. B. Morrison and J. McCapra, Nature (London) 192:774-776, 1961; K. Ogiuti, Jpn. J. Exp. Med. 14:19-28, 1936], high concentrations of salts, high concentrations of carbohydrates, high concentrations of low-molecular-weight alcohols, or the pressure of gyrase inhibitors. Under all these conditions, growth was necessary for the loss of motility. This loss of motility was correlated with a reduction in the amount of cellular flagellin. We isolated and studied mutants that are resistant to suppression of motility by some of these conditions, because of the ability to synthesize flagella under these conditions. The mutations were mapped to 42 min, a region of the chromosome where many of the flagellar genes map. We also studied the effect of a preexisting gyrA mutation which allowed flagellar formation in the presence of nalidixate.

Rational in vivo application of monoclonal antibodies for diagnosis and therapy of cancer requires an understanding of both the global and microscopic pharmacology of macromolecular ligands. Here, we introduce a new mathematical model for antibody distribution into small, prevascular, densely packed nodules (representing either primary or metastatic tumor). For the analysis, we link together several aspects of antibody pharmacology: the global (whole body) pharmacokinetics; transcapillary transport into normal tissue interstitium surrounding the nodule; diffusion into the nodule; nonspecific binding and/or partitioning; specific binding to tumor antigen; metabolism; and lymphatic outflow from the tissue space. Input parameter values are estimated from experimental studies in vitro, in animals, and in clinical trials. Our aim is to explore the sensitivity of antibody localization to variation in three of the important parameters of this model: the rate of transcapillary transport; the rate of lymphatic outflow; and the antigen density. Predictions based on this analysis include the following: (a) High rates of transcapillary transport influx or low rates of lymphatic efflux will enhance antibody percolation into the tumor nodule at early times after injection and increase the average antibody concentration in the tumor at all times; (b) Changes in antibody influx rate will affect the antibody distribution in the tumor at earlier times than do changes in the efflux rate; (c) Reducing the antigen concentration will increase the uniformity of antibody penetration but lower the average concentration in the tumor at all times after injection; and (d) Counter to intuition, lowering the antigen concentration can increase the peak concentrations achieved toward the center of the nodule. If, in addition, there is any metabolism of bound antibody, the concentration-time integral (i.e., the "area under the curve") for the center of the nodule will also be increased by decreasing the antigen concentration. These predictions directly reflect the "binding site barrier" hypothesis of Weinstein et al. (Ann. NY Acad. Sci., 507: 199-210, 1987) and Fujimori et al. (Cancer Res., 49:5656-5663, 1989; J. Nucl. Med., 31:1191-1198, 1990). In general, and perhaps surprisingly until one considers the problem carefully, the parameters governing antibody percolation can have opposite effects on the uniformity of antibody distribution at early and late times. These calculations, using the PERC program set, were done for antibodies, but we believe that the "binding site barrier" will also prove important for other injected macromolecules, for at least some highly bindable injected small molecules, for lymphokines and cytokines released from transfected cells injected in vivo, and, indeed, for endogenous species such as the autocrine-paracrine factors.

The activity of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin- 4(3H)-one (temozolomide) in the treatment of a panel of xenografts derived from ependymoma, medulloblastoma, and childhood and adult high-grade glioma was evaluated in athymic nude mice bearing s.c. and intracranial tumors. Temozolomide administered daily for a total of five doses demonstrated marked activity against a panel of Mer+ xenografts despite marginal to moderate activity of 1,3-bis(2-chloroethyl)-1-nitrosourea. The growth delays produced by temozolomide in these xenografts were 1.8-7.5-fold greater than those produced by procarbazine. Although temozolomide demonstrated marginal activity against the Mer+ cell line D341 Med when a 5-day schedule was used, a high-dose 1-day schedule resulted in moderate activity. Temozolomide produced increases in median survival of 1285% (adult glioma D-54 MG), 323% (childhood glioma D-456 MG), and 68% (ependymoma D612 EP). Pretreatment of mice with O6-benzylguanine increased temozolomide-induced mortality, requiring reduction of the dosage from 1200 to 750 mg/m2 on the single-day regimen. O6-Benzylguanine pretreatment of mice bearing Mer+ D341 Med increased the growth delay of temozolomide, in duplicate experiments, from -3.1 to 4.8 and 1.1 to 4.9 days. These studies suggest that temozolomide may be active in the treatment of a broad spectrum of central nervous system cancers, including Mer+ tumors resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea.

Dendritic cells (DC) can be present at distinct stages of differentiation within the immune system. Sallusto and colleagues have recently described an in vitro culture system suitable for analyzing the maturation processes of DC (Sallusto and colleagues, J. Exp. Med. 1994;179:1109-1118). Monocytes cultured for 6 d in the presence of granulocyte macrophage colony-stimulating factor and interleukin-4 develop into immature DC with a high endocytic capacity but a low capacity to stimulate T cells. When challenged by lipopolysaccharide, these cells upregulate costimulatory molecules, express CD83, and become mature DC. CCR1 and CCR5 chemokine receptors are highly expressed on immature DC and downregulated on mature DC. This in vitro system was used to characterize human lung DC. Lung DC were shown to express some characteristics of in vitro immature DC. These are: (1) low expression of the costimulatory molecules CD40, CD80, and CD86; (2) poor expression of the differentiation marker CD83 and no CD1a; and (3) good capacity to incorporate dextran. Lung DC express moderate levels of CCR1 and CCR5. However, lung DC, like in vitro mature DC, express high levels of major histocompatibility complex Class II molecules, show low expression of CD14 and CD64, and are characterized by their high capacity to stimulate allogeneic T cells to proliferate during mixed leukocyte reactions (MLRs). Although lung DC express low levels of CD80 and CD86, the important role of these costimulatory molecules in inducing high MLR was demonstrated by using blocking antibodies. Therefore, while lung DC have overall a phenotype and an endocytic capacity close to in vitro immature DC, they share, like in vitro mature DC, a powerful capacity to stimulate T cells.

Interleukin-1beta-converting enzyme (ICE, caspase-1) regulates key steps in inflammation and immunity, by activating the proinflammatory cytokines interleukin (IL-)1beta and IL-18, or mediating apoptotic processes. We recently provided evidence for the regulation of caspase-1 activity via an endogenous inhibitor expressed by human vascular smooth muscle cells (SMCs) (Schönbeck, U., M. Herzberg, A. Petersen, C. Wohlenberg, J. Gerdes, H.-D. Flad, and H. Loppnow. 1997. J. Exp. Med. 185:1287-1294). However, the molecular identity of this endogenous inhibitor remained undefined. We report here that the serine proteinase inhibitor (serpin) PI-9 accounts for the endogenous caspase-1 inhibitory activity in human SMCs and prevents processing of the enzyme's natural substrates, IL-1beta and IL-18 precursor. Treatment of SMC lysates with anti-PI-9 antibody abrogated the caspase-1 inhibitory activity and coprecipitated the enzyme, demonstrating protein-protein interaction. Furthermore, PI-9 antisense oligonucleotides coordinately reduced PI-9 expression and promoted IL-1beta release. Since SMCs comprise the majority of cells in the vascular wall, and because IL-1 is implicated in atherogenesis, we tested the biological validity of our in vitro findings within human atheroma in situ. The unaffected arterial wall contains abundant and homogeneously distributed PI-9. In human atherosclerotic lesions, however, PI-9 expression correlated inversely with immunoreactive IL-1beta, supporting a potential role of the endogenous caspase-1 inhibitor in this chronic inflammatory disease. Thus, our results provide new insights into the regulation of this enzyme involved in immune and inflammatory processes of chronic inflammatory diseases, and point to an endogenous antiinflammatory action of PI-9, dysregulated in a prevalent human disease.

Corticotropin-releasing factor (CRF) is one of the principle components of the stress response. The physiological effects of CRF are mediated by two receptor subtypes, CRF(1) and CRF(2). Recent data obtained with the selective CRF(2) antagonist antisauvagine-30 (ASV-30) has begun to suggest that both CRF receptor subtypes may play a role in stress-related behaviors. Exactly how these two receptor subtypes interact to modulate the behavioral and endocrine responses to stress is not clear, however. We have attempted to understand the role of the CRF(2) receptor in the behavioral and endocrine responses to stress by comparing the effects of ASV-30 with the mixed CRF(1)/CRF(2) receptor antagonist astressin. Centrally administered ASV-30 reduced anxiety-like behavior in BALB/c mice in three models of anxiety: marble burying [minimal effective dose (MED) = 3 nmol], open field (MED = 3 nmol), and elevated plus maze (MED = 0.1 nmol). ASV-30 did not change locomotor activity or the adrenocorticotropic hormone (ACTH) response to restraint stress. The potent mixed CRF(1)/CRF(2) antagonist astressin not only reduced anxiety-like behavior in all three models with equivalent potency but also blunted the ACTH response to restraint stress. Finally, the new selective CRF(2) receptor agonist urocortin-II produced a dose-dependent increase in anxiety-like behavior in the plus maze test. Therefore, our data suggest that the CRF(2) receptor plays a role in the behavioral, but not the hypothalamic-pituitary-adrenal axis, response to stress.

Identification of natural products capable of affording protection against UVB radiation-induced inflammatory responses and generation of oxidative stress may have important human health implications. The UVB exposure-induced skin injury and oxidative stress has been associated with a variety of skin disease conditions including photoaging, inflammation and cancer. Tea is a popular beverage consumed worldwide. In several mouse skin models, topical application as well as oral consumption of green tea has been shown to afford protection against chemical and UVB-induced carcinogenesis and inflammatory responses. In the present study, we investigated in human skin, whether topical application of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent in green tea, inhibits UVB-induced infiltration of leukocytes (macrophage/neutrophils), a potential source of generation of reactive oxygen species (ROS), and generation of prostaglandin (PG) metabolites. Human subjects were UVB irradiated on sun-protected skin to four times their minimal erythema dosage (MED) and skin biopsies or keratomes were obtained either 24 h or 48 h later. We found that topical application of EGCG (3 mg/2.5 cm2) before UVB (4 MED) exposure to human skin significantly blocked UVB-induced infiltration of leukocytes and reduced myeloperoxidase activity. These infiltrating leukocytes are considered to be the major source of generation of ROS. In the same set of experiments we found that topical application of EGCG before UVB exposure decreased UVB-induced erythema. In additional experiments, we found that microsomes from EGCG pretreated human skin and exposed to UVB, compared to UVB exposure alone, produced significantly reduced PG metabolites, particularly PGE2. The PG metabolites play a critical role in free radical generation and skin tumor promotion in multistage skin carcinogenesis. Careful microscopic examination of skin sections, stained with hematoxylin and eosin, under higher magnification (x400) also revealed that EGCG pretreated and UVB-exposed human skin contained fewer dead cells in the epidermis with comparison to nonpretreated UVB-exposed skin. Taken together, our data demonstrate that EGCG has the potential to block the UVB-induced infiltration of leukocytes and the subsequent generation of ROS in human skin. This may explain the possible mechanism involved in anti-inflammatory effects of green tea. We suggest that EGCG may be useful as a topical agent for protection against UVB-induced ROS-associated inflammatory dermatoses, photoaging and photocarcinogenesis. Further studies are warranted in this direction.