Gene therapy for sarcoma

Soft tissue sarcomas are mesenchymal tumors which respond poorly to systemic therapy. Recent studies suggest a higher response rate with an increased doxorubicin dosage. However, this was parallel with a profound hematotoxicity in 75% of patients. Transfer of the human multidrug resistance 1 (MDR1) gene to normal hematopoietic stem cells and transplantation may significantly reduce the hematotoxicity of anthracyclin-based chemotherapy. To test this concept of supportive gene therapy in advance of a clinical study, we transduced mobilized peripheral blood progenitor cells (PBPC) with the retroviral vector SF91m3 containing the human MDR1 gene, transplanted these cells to immune-deficient mice, allowed 6 weeks for engraftment to occur and treated the animals with MDR1-based chemotherapy. In the MDR1-transduced group the human leukocytes were significantly protected from the toxicity of chemotherapy (p < 0.05). While the gene transfer rate was in the range of 10% and thus comparable to recent clinical trials, the gene expression was 59% of transduced cells and thus significantly higher than previously reported for less-advanced vectors. On the other hand, ifosfamide, a drug which has been used successfully for stem cell mobilization, is active in soft tissue sarcoma. Due to these favorable characteristics sarcoma is an attractive target to test the efficacy of MDR1 gene therapy in a clinical setting. Gene therapeutic strategies may also be used to directly target sarcoma cells, e.g. by transfer of suicide genes. We found that adenoassociated virus 2 (AAV-2) vectors efficiently transduce human HS-1 and HT1080 sarcoma cells (>90%) while other tumor cell lines and primary human PBPC were less susceptible. The thymidine kinase (TK) suicide gene was cloned into an AAV-2 vector and a complete kill of TK-transduced HS-1 and HT1080 cells was observed following exposure to aciclovir or ganciclovir (GCV), while >90% of mock-transduced HS-1 cells survived at these dosages. Transplantation of those sarcoma cells to nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice resulted in a survival of >5 months in the AAV-TK-transduced/GCV-treated group, while the mice in the mock-transduced/GCV-treated group had died after 3 weeks. These data show that soft tissue sarcomas are a particularly suitable model system for the development and clinical testing of new gene therapeutic concepts.     

Arbovirus surveillance from 1990 to 1995 in the Barkedji area (Ferlo) of Senegal, a possible natural focus of Rift Valley fever virus

Surveillance for mosquito-borne viruses was conducted in Barkedji area from 1990 to 1995, following an outbreak of Rift Valley fever (RVF) virus in southern Mauritania. Mosquitoes, sand flies, and midges were collected from human bait and trapped by solid-state U.S. Army battery-powered CDC miniature light traps baited with dry ice or animals (sheep or chickens) at four ponds. Overall, 237,091 male and female mosquitoes representing 52 species in eight genera, 214,967 Phlebotomine sand flies, and 2,527 Culicoides were collected, identified, and tested for arboviruses in 9,490 pools (7,050 pools of female and 331 of male mosquitoes, 2,059 pools of sand flies and 50 pools of Culicoides). Viruses isolated included one Alphavirus, Babanki (BBK); six Flaviviruses, Bagaza (BAG), Ar D 65239, Wesselsbron (WSL), West Nile (WN), Koutango (KOU), Saboya (SAB); two Bunyavirus, Bunyamwera (BUN) and Ngari (NRI); two Phleboviruses, Rift Valley fever (RVF) and Gabek Forest (GF); one Orbivirus, Ar D 66707 (Sanar); one Rhabdovirus, Chandipura (CHP); and one unclassified virus, Ar D 95537. Based on repeated isolations, high field infection rates and abundance, Culex appeared to be the vectors of BAG, BBK, Ar D 65239 (BAG-like), and WN viruses, Ae. vexans and Ae. ochraceus of RVF virus, Mansonia of WN and BAG viruses, Mimomyia of WN and BAG viruses, and Phlebotomine of SAB, CHP, Ar D 95537, and GF viruses. Our data indicate that RVF virus circulated repeatedly in the Barkedji area.     

Circulation of the poliovirus in endemic zones with children vaccinated by the oral polio vaccine

Strategies aiming to eradicate the poliovirus and poliomyelitis seek primarily to eliminate wild strains associated with the disease, by means of world wide vaccination campaigns using the oral attenuated vaccine (OPV). OPV contains attenuated viral strains which retain their replicating capacity in the digestive tract and thus induce the development of an antiviral local intestinal immunity and limit the circulation of the virus. In such a context, poliomyelitis surveillance laboratories should study above all cases of acute flaccid paralysis (AFP), highlighting the circulation of wild strains, identifying regional reservoirs and guiding vaccination strategies. Alongside circulation, there appear to be important genetic and phenotypic shifts in vaccinating strains, since the OPV is capable of preserving a reservoir of pathogenic stains and thereby impairing vaccination efficacy and the eradication of the virus. Furthermore, non-polio enteroviruses should be considered as a source of emerging pathogenic strains. These questions are being studied by the Pasteur Institute with the objective of determining the effects of OPV campaigns on the circulation of the poliovirus. We have studied the poliovirus vaccine and the circulation of wild strains in urban and peripheral urban areas in African countries known to be endemic for poliomyelitis (Central African Republic, Madagascar, C?te d'Ivoire). The study population consisted of children who had already been vaccinated and new-borns in the course of vaccination. We also evaluated the diffusion of the vaccine strains in their immediate environment. Genetic interchanges were taken into account. For children who received the 3-4 OPV doses, asymptomatic virus excretion was insignificant (0.4-2.4%). The rate of virus excretion in the surrounding environment of children in the course of being vaccinated was relatively low (1.76-5.3%). Our study also detected variant and recombinant strains.     

PCR assays for identification of Coccidioides posadasii based on the nucleotide sequence of the antigen 2/proline-rich antigen

A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens.     

Lipopolysaccharide contamination of beta-lactoglobulin affects the immune response against intraperitoneally and orally administered antigen

BACKGROUND: Microbial components in the environment are potent activators of the immune system with capacity to shift the active immune response towards priming of Th1 and/or Th2 cells. Lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria, is extensively present in food products like cow's milk. It is not well established, however, how this presence of LPS affects oral tolerance induction. METHODS: We studied the effect of LPS contamination in a commercial preparation of the cow milk protein beta-lactoglobulin (beta-LG) on antigen-specific immune responses. IgG1/IgG2a production upon intraperitoneal immunization without adjuvant was measured, and oral tolerance induction against beta-LG after administration of either an aqueous solution or water-in-oil (w/o) emulsion of beta-LG was evaluated. RESULTS: LPS contamination of beta-LG provoked a beta-LG-specific IgG2a response, as well as an enhanced beta-LG-specific IgG1 response upon intraperitoneal immunization. Oral tolerance induction to beta-LG was induced by aqueous solutions of beta-LG with and without LPS administration. Conversely, oral administration of w/o-emulsified beta-LG prevented oral tolerance to beta-LG only when the beta-LG was contaminated with LPS. CONCLUSIONS: LPS contamination of an aqueous protein solution does not affect oral tolerance induction, whereas LPS present in emulsion prevents oral tolerance induction towards the food protein.     

Pathology and pathogenesis of bioterrorism-related inhalational anthrax

During October and November 2001, public health authorities investigated 11 patients with inhalational anthrax related to a bioterrorism attack in the United States. Formalin-fixed samples from 8 patients were available for pathological and immunohistochemical (IHC) study using monoclonal antibodies against the Bacillus anthracis cell wall and capsule. Prominent serosanguinous pleural effusions and hemorrhagic mediastinitis were found in 5 patients who died. Pulmonary infiltrates seen on chest radiographs corresponded to intraalveolar edema and hyaline membranes. IHC assays demonstrated abundant intra- and extracellular bacilli, bacillary fragments, and granular antigen-staining in mediastinal lymph nodes, surrounding soft tissues, and pleura. IHC staining in lung, liver, spleen, and intestine was present primarily inside blood vessels and sinusoids. Gram's staining of tissues was not consistently positive. In 3 surviving patients, IHC of pleural samples demonstrated abundant granular antigen-staining and rare bacilli while transbronchial biopsies showed granular antigen-staining in interstitial cells. In surviving patients, bacilli were not observed with gram's stains. Pathological and IHC studies of patients who died of bioterrorism-related inhalational anthrax confirmed the route of infection. IHC was indispensable for diagnosis of surviving anthrax cases. The presence of B. anthracis antigens in the pleurae could explain the prominent and persistent hemorrhagic pleural effusions.     

TK-GFP fusion gene virus vectors as tools for studying the features of HSV-TK/ganciclovir cancer gene therapy in vivo

The fusion gene of herpes simplex virus thymidine kinase and green fluorescent protein (TK-GFP) was shown to be a versatile tool for examining the features of thymidine kinase/ganciclovir gene therapy in vitro. In this study, we used viral vectors carrying the fusion gene to characterize the aspects of this gene therapy form in rodent tumor models. Growth of subcutaneous 9L rat tumors transduced ex vivo with TK-GFP gene was prevented when ganciclovir (GCV) treatment was initiated immediately after tumor inoculation. Established tumors (>100 mm(3)), however, were untreatable despite the initial 55% proportion of TK-GFP positive cells. This was due to a rapid clearance of TK-GFP positive cells, but not GFP positive cells. Propidium iodide staining revealed that TK-GFP lentivirus vector was able to induce apoptosis/necrosis in 9L cells, as opposed to the respective GFP vector. Furthermore, when a subcutaneous nude mouse tumor model was used, the percentage of TK-GFP positive cells in vivo was maintained similarly as in cultured cells, suggesting contribution of a fully functional immune response to the disappearance of fusion gene positive cells. In vivo gene transfer studies: adenovirus TK-GFP vector injections resulted in about 25% gene transfer efficiency to 9L tumors and showed that their growth could be significantly reduced even when the tumor volumes were already >120 mm(3). Part of the effect was shown to be due to cytotoxicity of the vector. In summary, our results demonstrate the utility of TK-GFP fusion gene-carrying viral vectors in animal studies and show that readily detectable therapeutic genes can help us to understand the complicated nature of in vivo cancer gene therapy experiments.