Apoptosis as a target for gene therapy in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages and plasma cells, all of which manifest signs of activation. All these cells proliferate abnormally, invade bone and cartilage, produce an elevated amount of pro-inflammatory cytokines, metalloproteinases and trigger osteoclast formation and activation. Some of the pathophysiological consequences of the disease may be explained by the inadequate apoptosis, which may promote the survival of autoreactive T cells, macrophages or synovial fibroblasts. Although RA does not result from single genetic mutations, elucidation of the molecular mechanisms implicated in joint destruction has revealed novel targets for gene therapy. Gene transfer strategies include inhibition of pro-inflammatory cytokines, blockade of cartilage-degrading metalloproteinases, inhibition of synovial cell activation and manipulation of the Th1-Th2 cytokine balance. Recent findings have iluminated the idea that induction of apoptosis in the rheumatoid joint can be also used to gain therapeutic advantage in the disease. In the present review we will discuss different strategies used for gene transfer in RA and chronic inflammation. Particularly, we will highlight the importance of programmed cell death as a novel target for gene therapy using endogenous biological mediators, such as galectin-1, a beta-galactoside-binding protein that induces apoptosis of activated T cells and immature thymocytes.

An Adenovirus Vector Containing the Suicide Gene Thymidine Kinase for a Broad Application in Cancer Gene Therapy

Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK) gene, followed by administration of the antiviral drug ganciclovir (GCV). The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK) driven by three strong promoters (P CMV IE, SV40 and EN1) and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.

Schistosoma mansoni heat shock protein 70 elicits an early humoral immune response in S. mansoni infected baboons

A study was undertaken to search for DNA recombinant Schistosoma mansoni proteins responsible for eliciting an antibody response from the host at a very early phase after infection. A S. mansoni adult worm cDNA expression library was screened using pooled sera from baboons with four weeks of infection. Based on their specific reactivity with the S. mansoni infected sera and no reactivity when tested against the pre-infection sera from the same baboons, four clones were selected for further studies. Sequence analysis revealed that they were homologous to the S. mansoni heat shock protein 70 (hsp70). The insert sizes of the four selected clones varied from 1150 to 2006 bp. The preliminary characterization for antibody reactivity against a panel of baboon sera showed that the longest clone was the most reactive, eight out of eight acute and three out of four chronic sera reacting positively to this clone. The shortest clone was the least reactive. Our results suggest that the S. mansoni hsp70 elicits an early and strong antibody response in baboons and that antibodies to this protein can be detected in chronically infected animals. Therefore S. mansoni hsp70 may be a valid target for immunodiagnosis. However further studies are needed to identify the portion of the hsp70 that best fits the requirements for a valuable diagnostic antigen.

Laboratory evaluation of methanolic extract of Atlantia monophylla (Family: Rutaceae) against immature stages of mosquitoes and non-target organisms

Methanolic extracts of the leaves of Atlantia monophylla (Rutaceae) were evaluated for mosquitocidal activity against immature stages of three mosquito species, Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti in the laboratory.Larvae of Cx. quinquefasciatus and pupae of An. stephensi were found more susceptible, with LC50 values of 0.14 mg/l and 0.05 mg/l, respectively. Insect growth regulating activity of this extract was more pronounced against Ae. aegypti, with EI50 value 0.002 mg/l. The extract was found safe to aquatic mosquito predators Gambusia affinis, Poecilia reticulata, and Diplonychus indicus, with the respective LC50 values of 23.4, 21.3, and 5.7 mg/l. The results indicate that the mosquitocidal effects of the extract of this plant were comparable to neem extract and certain synthetic chemical larvicides like fenthion, methoprene, etc.