Control of the cell survival/death decision by cannabinoids

Cannabinoids, the active components of Cannabis sativa (marijuana), and their derivatives produce a wide spectrum of central and peripheral effects, some of which may have clinical application. The discovery of specific cannabinoid receptors and a family of endogenous ligands of those receptors has attracted much attention to cannabinoids in recent years. One of the most exciting and promising areas of current cannabinoid research is the ability of these compounds to control the cell survival/death decision. Thus cannabinoids may induce proliferation, growth arrest, or apoptosis in a number of cells, including neurons, lymphocytes, and various transformed neural and nonneural cells. The variation in drug effects may depend on experimental factors such as drug concentration, timing of drug delivery, and type of cell examined. Regarding the central nervous system, most of the experimental evidence indicates that cannabinoids may protect neurons from toxic insults such as glutamaergic overstimulation, ischemia and oxidative damage. In contrast, cannabinoids induce apoptosis of glioma cells in culture and regression of malignant gliomas in vivo. Breast and prostate cancer cells are also sensitive to cannabinoid-induced antiproliferation. Regarding the immune system, low doses of cannabinoids may enhance cell proliferation, whereas high doses of cannabinoids usually induce growth arrest or apoptosis. The neuroprotective effect of cannabinoids may have potential clinical relevance for the treatment of neurodegenerative disorders such as multiple sclerosis, Parkinson's disease, and ischemia/stroke, whereas their growth-inhibiting action on transformed cells might be useful for the management of malignant brain tumors. Ongoing investigation is in search for cannabinoid-based therapeutic strategies devoid of nondesired psychotropic effects.     

Mutational analysis of the yeast multidrug resistance ABC transporter Pdr5p with altered drug specificity

Multidrug resistance ABC transporter Pdr5p of Saccharomyces cerevisiae is particularly important due to its ability to export a wide range of unrelated substrates. To clarify its function, we generated Pdr5p mutants by random mutagenesis and screened for mutants with altered drug specificity in vivo by using 5 drug compounds. Nine point mutations that caused significant changes in drug specificity distributed throughout the length of Pdr5p, namely, in the extracellular, transmembrane or cytoplasmic regions of the transporter. We then investigated their effects upon drug resistance, using 36 chemically related or distinct substrates. From this study, overall geometry of the Pdr5p was suggested to contribute in acquiring the enormous range of drug specificity. Based on their ability to inhibit the growth of the mutant strains, the 36 tested drugs were classified into: drugs to which the mutants responded differently (Group 1), drugs to which all the mutants showed sensitivity (Group 2), and drugs to which all the mutants exhibited resistance (Group 3). The ability of the compounds to be partitioned to the plasma membrane seemed an important factor for recognition by Pdr5p.     

Juvenile sudden death in a family with polymorphic ventricular arrhythmias caused by a novel RyR2 gene mutation: evidence of specific morphological substrates

We report on a family with a history of sudden death and effort-induced polymorphic ventricular arrhythmias. The index case was a 17-year-old boy who died suddenly and at postmortem had evidence of fibrofatty replacement in the right ventricular free wall, consistent with arrhythmogenic right ventricular cardiomyopathy, as well as calcium phosphate deposits within the myocytes. A molecular genetics investigation carried out in the paraffin-embedded myocardium of the subject and in blood samples of family members disclosed a missense mutation in exon 3 (230C-->T; A77V) of the cardiac ryanodine receptor type 2 gene. The carriers showed effort-induced polymorphic ventricular tachycardia in the setting of normal resting electrocardiogram and trivial echocardiographic abnormalities, consistent with catecholaminergic polymorphic ventricular tachycardia. The observation of both arrhythmogenic right ventricular cardiomyopathy type 2 and catecholaminergic polymorphic ventricular tachycardia in the same family suggests that the two entities might correspond to different degrees of phenotypic expression of the same disease. This experience underscores the importance of a precise autopsy diagnosis in the case of sudden cardiac death, including molecular genetics, and the mission of pathologists to guide further clinical investigation of family members.     

Mechanical heart valve prostheses:: identification and evaluation (erratum)

Mechanical heart value prostheses have been in use since the 1950s. Many prostheses have been used for a while and then discontinued. Today, there are a large number and variety of prostheses in use and an even larger variety that are in place in patients. These may be explanted at any time for a number of reasons. It is essential for the practicing pathologist to be able to identify the prosthesis and be aware of some of its reported complications and modes of failure. This article, and a second one on bioprosthetic heart valves, is designed as a ready reference guide to heart valve prostheses, their important identifying features, their common complications, and modes of failure. It should help in the accurate identification of explanted prosthetic valves and more definitive reports. This accuracy of identification as well as tracking of abnormalities noted will, we hope, permit the identification of new failure modes and the recording of causes of failure of new (or even modified) prosthetic heart valves.     

Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and genotype Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with Mycobacterial strains

The performance of two DNA line probe assays, a new version of INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium) and the GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), were evaluated for identification of mycobacterial species isolated from liquid cultures. Both tests are based on a PCR technique and designed for simultaneous identification of different mycobacterial species by reverse hybridization and line probe technology. The INNO-LiPA Mycobacteria v2 targeting the 16S-23S rRNA gene spacer region was developed for the simultaneous identification of 16 different mycobacterial species. The GenoType Mycobacterium, which targets the 23S rRNA gene, allows simultaneous identification of 13 mycobacterial species. Both tests were evaluated on 110 mycobacterial strains belonging to 22 different mycobacterial species (20 reference strains, 83 clinical strains, and 4 Mycobacterium kansasii strains isolated from tap water) that were previously inoculated into MB/BacT bottles. The sensitivity of both methods, defined as the number of positive results obtained with the Mycobacterium genus probe together with an interpretable result on the number of samples tested was 110 of 110 (100%) for INNO-LiPA and 102 of 110 (92.7%) for GenoType. For samples with interpretable results, INNO-LiPA was able to correctly identify 109 of 110 samples (99.1%), whereas the GenoType correctly identified 100 of 102 samples (98.0%). Both tests were easy to perform, rapid, and reliable when applied to mycobacterial identification directly from MB/BacT bottles.     

Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system

Optimization of three enzyme immunoassays of very high sensitivity using three antiprolamin monoclonal antibodies (MAbs) (13B4, 11C4 and 12A1) is presented here. These MAbs are specific for those prolamins toxic for coeliac patients, as determined by immunoblotting analysis. Biotinylated MAbs were used in two of the assays. In a competitive ELISA, the binding of each biotinylated MAb to a gliadin‐coated solid phase was inhibited by gliadin in the fluid phase. The best result was obtained using the biotinylated MAbl3B4 (detection limit: 20 ng ml^?1). With regard to capture ELISA, we tested the performance of the three MAbs. In this sandwich ELISA, the MAb used for antigenic capture was the same as that used as secondary biotinylated antibody. The MAbl2Al had the best performance (detection limit: 1 ng ml^?1). The use of biotin‐labelled gliadin in a quantitative immunoassay with a detection limit of 5 ng ml^?1 is also reported. This assay involves an antigenic capture using the MAbl2Al followed by a competition between biotinylated and non‐biotinylated gliadin. We have found the use of the streptavidin‐biotin interaction as signal amplification system to be very useful. This technique, as far as we know, has not been previously reported for gliadin quantification.     

Analysis of IFN-kappa expression in pathologic skin conditions: downregulation in psoriasis and atopic dermatitis.

Interferon-kappa (IFN-kappa) is a type I IFN expressed by keratinocytes, monocytes and dendritic cells (DCs). In human keratinocytes, it is produced in response to double-stranded RNA (dsRNA) and other IFNs and protects from viral infections. In monocytes and DCs, IFN-kappa induces tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) and inhibits lipopolysaccharide (LPS)-induced IL-12. In this study, we evaluated IFN-kappa expression in skin lesions of patients with common immune-mediated inflammatory disorders using immunohistochemical techniques. IFN-kappa was not detectable in healthy skin but was strongly expressed in allergic contact dermatitis and lichen planus-affected skin. IFN-kappa was localized mainly in basal and suprabasal keratinocytes and in some leukocytes infiltrating the dermis. In contrast, IFN-kappa expression in psoriatic or atopic dermatitis (AD) pidermis was weak and detectable in only 2 of 5 patients examined. Consistently, cultured keratinocytes and monocytes obtained from psoriatic and AD patients expressed null or low levels of IFN-kappa in response to IFN-gamma, which strongly upregulates IFN-kappa in normal keratinocytes. IFN-kappa accumulated in keratinocyte cytoplasm and plasma membrane, and only limited amounts were released extracellularly. Soluble IFN-kappa did not influence keratinocyte proliferation or chemokine and membrane molecule expression, and only its membrane-associated form activated IFN-stimulated response element (ISRE) signaling. Given the difference in IFN-kappa expression levels in the skin disorders examined, IFN-kappa presence or deficiency might have different pathogenetic consequences depending also on other disease-specific intrinsic alterations.     

Isolation of protective antigens from Trypanosoma lewisi by using trypanostatic (ablastic) immunoglobulin G from the surface coat.

Antigens were purified from extracts of Trypanosoma lewisi on immunoadsorbent columns of trypanostatic immunoglobulin G eluted from parasite surface coats at 8 days postinfection. Eight absorbed proteins, with molecular weights between 15,000 and 70,000, were identified. These surface coat antigens (SCAgs) were then used to immunize rats. After immunization, sera were assayed in vitro for levels of circulating trypanostatic and trypanocidal antibodies. Approximately half of the immune sera had higher levels of trypanostatic antibody, compared with control sera; no trypanocidal antibodies (agglutinins) were detected in any of the sera. The rats were then challenged intraperitonally, and the parasitemias and division rates of the parasites were monitored. Parasitemias of all immunized rats were significantly (P less than 0.01) lower and of shorter duration than those of the controls. Division rates of trypanosomes were also significantly (P less than 0.01) lower in all immunized rats at all times before total cessation of division compared with control rats. A clear dose-response effect was observed, with greater amounts of SCAg eliciting higher levels of protection. Purified SCAgs were also more effective immunogens than were the crude trypanosome extracts from which they had been purified, and in which other proteins in addition to the SCAgs were present. These data provide conclusive evidence that the immunoglobulin G in the surface coats of T. lewisi, adsorbed during the course of infection, is specific antibody, in that it can be used to isolate parasite antigens that elicit a trypanostatic response in rats immunized with them.     

Herpes simplex virus type 1 can either suppress or enhance human immunodeficiency virus type 1 replication in CD4-positive T lymphocytes

It was proposed recently that CEM CD4-positive T cells infected chronically by herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1) (CEM(HSV/HIV)) may be used as a model for studying HIV/HSV interactions. To ascertain whether HSV-HIV coinfection of T lymphocytes has a role in promoting progression of lentiviral infection, T cells infected chronically by either HSV-1 (CEM(HSV)) or HIV-1 (CEM(HIV)) were challenged with a superinfecting dose of HIV-1 or HSV-1. The results show a positive influence on HIV growth when CEM(HIV) cells were superinfected with HSV-1 to an extent that was dependent on the multiplicity of superinfection used. In contrast, HIV superinfection of CEM(HSV) cells resulted in a delay of HIV-1 production and in a lack of HSV-mediated LTR transactivation. These effects were due to cell growth inhibition and apoptosis, resulting from persistent HSV-1 infection. Treatment of CEM(HSV) with acyclovir inhibited completely the HSV-1 cytopathic effects and allowed efficient HIV-1 replication. These data may be relevant in clarifying the role of HIV/HSV interaction in the pathogenesis of AIDS.