Infectivity inhibition of HIV prototype strains by antibodies directed against a peptide sequence of mycoplasma

Antibodies prepared against a peptide corresponding to the site of cyto-adherence of Mycoplasma genitalium adhesine inhibit or reduce the infectivity of the HIV-1BRU and HIV-2ROD strains of Human Immunodeficiency Virus in lymphoid cells. These results strengthen the hypothesis that some mycoplasmas may play an important part in HIV replication and pathogenicity.     

Efficient removal of LoxP-flanked genes by electroporation of Cre-recombinase mRNA

Introduction of Cre-recombinase in target cells is currently achieved by transfection of plasmid DNA or by viral-mediated transduction. However, efficiency of non-viral DNA transfection is often low in many cell types, and the use of viral vectors for transduction implies a more complex and laborious manipulation associated with safety issues. We have developed a non-viral non-DNA technique for rapid and highly efficient excision of LoxP-flanked DNA sequences based on electroporation of in vitro transcribed mRNA encoding Cre-recombinase. A K562-DSRed[EGFP] cell line was developed in order to measure Cre-mediated recombination by flow cytometric analysis. These cells have a stable integrated DSRed reporter gene flanked by two LoxP sites, and an EGFP reporter gene, which could only be transcribed when the coding sequence for DSRed was removed. The presented data show recombination efficiencies, as measured by appearance of EGFP-fluorescence, of up to 85% in Cre-recombinase mRNA-electroporated K562-DSRed[EGFP] cells. In conclusion, mRNA electroporation of Cre-recombinase is a powerful, safe, and clinically applicable alternative to current technologies used for excision of stably integrated LoxP-flanked DNA sequences.     

Incorporation of tritiated thymidine into DNA of rat liver: a critique of various evaluation methods]

We have measured the incorporation of 3H-(methyl)-thymidine by cell cultures of rat foetal liver and in vivo by the livers of young rats stimulated by casein, in order to compare three methods for the extraction of DNA. The DNA was extracted by three different techniques: perchloric acid precipitation, trichloroacetic acid precipitation and phenol extraction, and its specific activity was determined. The radioactive labelling was also determined for the lipid, ribonucleic acid and protein fractions for the two first methods, in both of which 70 p. cent of the incorporated tritium is found in the DNA fraction and about 10 p. cent in each of the other fractions. The determination of the specific radioactivity of DNA gives similar results for the three extraction methods. However, since larger yields were obtained by both acid precipitation techniques than by phenol extraction, we believe them to be more suitable for studies on cell cultures.     

Dendritic cell vaccination in acute myeloid leukemia

The prognosis of patients with acute myeloid leukemia (AML) remains dismal, with a 5-year overall survival rate of only 5.2% for the continuously growing subgroup of AML patients older than 65 years. These patients are generally not considered eligible for intensive chemotherapy and/or allogeneic hematopoietic stem cell transplantation because of high treatment-related morbidity and mortality, emphasizing the need for novel, less toxic, treatment alternatives. It is within this context that immunotherapy has gained attention in recent years. In this review, we focus on the use of dendritic cell (DC) vaccines for immunotherapy of AML. DC are central orchestrators of the immune system, bridging innate and adaptive immunity and critical to the induction of anti-leukemic immunity. We discuss the rationale and basic principles of DC-based therapy for AML and review the clinical experience that has been obtained so far with this form of immunotherapy for patients with AML.     

Combined Approaches for Drug Design Points the Way to Novel Proline Racemase Inhibitor Candidates to Fight Chagas’ Disease

Chagas’ disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.     

Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for 'proline racemase' virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not 'one way' directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on constraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host.     

Flow cytometric analysis of T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia: correlation with clinical stage

Several authors have studied the T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia (B-CLL), but previous studies were performed after preceding enrichment procedures, which are known to cause selective losses of certain subpopulations. To correct for this deficiency we used flow cytometric analysis, which enabled us to measure subpopulations directly on total blood samples. We studied T-lymphocyte subsets with OKT monoclonal antibodies in 45 patients with B-CLL. Serum levels of IgG, IgA and IgM were assayed simultaneously and findings were correlated with clinical stage (Rai classification). The absolute number of CD4-positive cells decreased in more advanced Rai stages, while the absolute number of CD8-positive cells increased, resulting in a progressive reduction in CD4/8 ratio. Results from patients in stages with equal prognosis (Rai I and II, Rai III and IV) were similar and when these results were grouped the observed differences were highly significant and clearly correlated with all prognostic groups.     

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

Background

Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs.

Results

Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons.

Conclusion

We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.     

Interleukin-15-Induced CD56+ Myeloid Dendritic Cells Combine Potent Tumor Antigen Presentation with Direct Tumoricidal Potential

Dendritic cells (DCs) are the quintessential antigen-presenting cells of the human immune system and play a prime role in coordinating innate and adaptive immune responses, explaining the strong and still growing interest in their application for cancer immunotherapy. Much current research in the field of DC-based immunotherapy focuses on optimizing the culture conditions for in vitro DC generation in order to assure that DCs with the best possible immunogenic qualities are being used for immunotherapy. In this context, monocyte-derived DCs that are alternatively induced by interleukin-15 (IL-15 DCs) have attracted recent attention due to their superior immunostimulatory characteristics. In this study, we show that IL-15 DCs, in addition to potent tumor antigen-presenting function, possess tumoricidal potential and thus qualify for the designation of killer DCs. Notwithstanding marked expression of the natural killer (NK) cell marker CD56 on a subset of IL-15 DCs, we found no evidence of a further phenotypic overlap between IL-15 DCs and NK cells. Allostimulation and antigen presentation assays confirmed that IL-15 DCs should be regarded as bona fide myeloid DCs not only from the phenotypic but also from the functional point of view. Concerning their cytotoxic activity, we demonstrate that IL-15 DCs are able to induce apoptotic cell death of the human K562 tumor cell line, while sparing tumor antigen-specific T cells. The cytotoxicity of IL-15 DCs is predominantly mediated by granzyme B and, to a small extent, by tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL) but is independent of perforin, Fas ligand and TNF-α. In conclusion, our data provide evidence of a previously unappreciated role for IL-15 in the differentiation of human monocytes towards killer DCs. The observation that IL-15 DCs have killer DC capacity lends further support to their implementation in DC-based immunotherapy protocols.