Debra Swanson

Diagnosis of proliferative enteritis in frozen and formalin-fixed, paraffin-embedded tissues from a hamster, horse, deer and ostrich using a Lawsonia intracellularis-specific multiplex PCR assay.

Proliferative enteritis (PE) is an enteric disease that has been reported in a variety of animals. It is caused by an obligate intracellular bacterium identified in swine as Lawsonia intracellularis. The organism can be detected ante-mortem in swine with PE using molecular diagnostic methods. The disease can be diagnosed post-mortem in all species by gross examination of tissues and special histologic staining procedures. In this study we extracted total DNA from frozen or formalin-fixed, paraffin-embedded tissues from cases of pig, hamster, horse, deer and ostrich PE. The samples were subjected to a multiplex PCR reaction using primers specific for a swine isolate of L. intracellularis. Identical sized PCR products were detected in samples from all animals with PE and the specificity of the PCR reaction for L. intracellularis was demonstrated by Southern-blotting and hybridization using specific probes. These results suggest that the intracellular organism of PE in these species are all very closely related to the causative agent of PE in swine, L. intracellularis. In addition, this multiplex PCR assay can be used to detect the organism in frozen or archival tissues, facilitating retrospective diagnosis of PE.

Comparison of the 16S ribosomal DNA sequences from the intracellular agents of proliferative enteritis in a hamster, deer, and ostrich with the sequence of a porcine isolate of Lawsonia intracellularis

Proliferative enteritis is an enteric disease that affects a variety of animals. The causative agent in swine has been determined to be an obligate intracellular bacterium, Lawsonia intracellularis, related to the sulfate-reducing bacterium Desulfovibrio desulfuricans. The intracellular agents found in the lesions of different animal species are antigenically similar. In addition, strains from the pig, ferret, and hamster have been shown to be genetically similar. In this study we performed a partial 16S ribosomal DNA sequence analysis on the intracellular agent of proliferative enteritis from a hamster, a deer, and an ostrich and compared these sequences to that of the porcine L. intracellularis isolate. Results of this study indicate that the intracellular agents from these species with proliferative enteritis have high sequence similarity, indicating that they are all in the genus Lawsonia and that they may also be the same species, L. intracellularis.

Cytotoxicity Associated with Artemis Overexpression After Lentiviral Vector-Mediated Gene Transfer

Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A.

Protection of Mice from Methotrexate Toxicity by ex Vivo Transduction Using Lentivirus Vectors Expressing Drug-Resistant Dihydrofolate Reductase

Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vector-modified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.

In vivo protection of activated Tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate.

Nonmyeloablative allogeneic hematopoietic stem cell (HSC) transplantation can cure malignant and nonmalignant diseases affecting the hematopoietic system, such as severe combined immunodeficiencies, aplastic anemia and hemoglobinopathies. Although nonmyeloablative is favored over myeloablative transplantation for many patients, graft rejection remains problematic. One strategy for decreasing rejection is to protect donor activated T cells in the graft from methotrexate (MTX) by genetically modifying the cells to express MTX‐resistant dihydrofolate reductase (Tyr22‐DHFR), leaving the immunosuppressive effects of MTX to act solely on activated host T lymphocytes, shifting the balance to favor allogeneic engraftment.

773. Chemoprotection of Mouse Marrow Transplant Recipients by Ex Vivo Lentiviral Transduction of Murine tyr22 Dihydrofolate Reductase Conferring Resistance to Methotrexate

Top of pageAbstract Methotrexate (MTX) impedes tumor development by inhibiting dihydrofolate reductase (DHFR), an enzyme that catalyzes the conversion of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate (THF). MTX depletes cells of THF, a required precursor for de novo purine and thymidine nucleotide synthesis, and has toxic effects on normal cells of the hematopoietic and gastrointestinal systems. To decrease MTX sensitivity, we introduced a MTX-resistant murine DHFR variant into mouse bone marrow by lentiviral transduction, so as to efficiently transduce non-dividing hematopoietic stem cells. We constructed HIV-1-based lentiviral vectors containing an enhanced green fluorescent protein (eGFP) gene with or without a murine tyr22 DHFR cDNA under transcriptional control of the human EF1-|[alpha]| promoter. These VSV-G-protein pseudotyped lentiviral vectors contained a self-inactivating 3|[prime]| LTR, rev response element (RRE) and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to enhance transduction efficiency. Viral vector was produced by co-transfection in 293T cells and concentrated 100-fold by centrifugation to a titer of 1 x 108 IU/ml, as determined by MTX-resistant colony formation and by flow cytometry for GFP expression. We transduced unfractionated bone marrow from normal Bl/6 Ly5.1 mice with either DHFR-GFP or GFP virus for 12 hours at a multiplicity of infection of 10 in the presence of cytokines (mSCF, IL-6, IL-3). Congenic Ly5.2 recipients received sub-lethal irradiation (700 cGy) prior to transplantation of 3 x 106 transduced marrow cells. MTX or PBS were administered daily starting twenty- four hours after transplantation in three recipient cohorts. Animals that received DHFR-virus transduced marrow recovered to normal hematocrit levels by week 3 in spite of MTX administration (44.7% +/- 2.5). In contrast, animals that received GFP-transduced marrow with subsequent MTX administration exhibited extremely reduced hematocrits (16.5% +/- 0.5) at week 3 and did not survive MTX dose-escalation to 2 mg/kg/day. Animals on MTX therapy that received DHFR-GFP transduced marrow exhibited increased donor cell (Ly5.1) engraftment and significant GFP marking in lymphoid (CD3, B220) and myeloid (GR-1) populations, while recipients that received GFP-transduced marrow exhibited lower levels of peripheral blood mononuclear cells and GFP+ leukocytes. Studies are currently underway to determine transgene copy number in repopulated marrow cells and assess MTX toxicity for the gastrointestinal tract. We conclude that transplantation of lentivirally transduced MTXr-DHFR transgenic marrow supports bone marrow repopulation during MTX chemotherapy in irradiated recipients, providing significant chemoprotection that permits MTX dose-escalation. These pre-clinical results support the development of a clinical gene therapy protocol to provide chemoprotection by lentiviral gene transfer.

279. Methotrexate Chemotherapy of L1210 Leukemia in Mice Transplanted with Transgenic Marrow Expressing Drug-Resistant Dihydrofolate Reductase

Methotrexate (MTX) is an effective chemotherapeutic agent in the treatment of several proliferative tumors, most notably acute lymphocytic leukemia, Ewings sarcoma and osteosarcoma. However, methotrexate also has considerable toxicity for normal proliferative tissues, including hematopoietic cells and cells of the gastrointestinal tract. The toxicity of methotrexate thus limits its chemotherapeutic effectiveness. MTX acts by binding to dihydrofolate reductase (DHFR) as a competitive inhibitor of the enzyme. We are investigating the possibility of expressing drug-resistant forms of DHFR in hematopoietic cells as a means of protecting the recipient from the toxicity associated with MTX chemotherapy. We previously demonstrated substantial protection of recipient mice from lethal toxicity of MTX by transplantation of transgenic marrow expressing Arg22 or Tyr22 forms of drug-resistant DHFR. Here we report that the increased MTX dose tolerance afforded by transplantation with marrow expressing the tyr22 form of drug-resistant DHFR allows for improved chemotherapy of murine L1210 leukemia. In our experimental model, animals were administered a subcutaneous dose of 10e6 L1210 tumor cells. Three days later, the animals were given a sublethal (650 cGy) dose of Cs irradiation. Our preliminary studies demonstrated that this course of treatments left a consistent amount of residual disease in the animals. One day after irradiation, animals were transplanted with 107 bone marrow cells flushed either from normal animals or from Tyr22 DHFR transgenic animals, and on the following day MTX administration was initiated. Control mice administered PBS developed tumor at a median of 12 days after subcutaneous injection of L1210 cells. Administration of 4 mg/kg MTX resulted in a considerable delay in the emergence of tumor for groups given either normal BMT or DHFR transgenic BMT. However, animals transplanted with normal marrow suffered from MTX toxicity, evidenced by reduced hematocrit, while there was no anemia observed in animals transplanted with DHFR transgenic marrow. A second experiment was conducted in the same way except that MTX was administered at a higher dose of 10 mg/kg/day. In this experiment, all control animals transplanted with normal marrow succumbed to toxicity associated with drug administration, while animals transplanted with DHFR transgenic marrow were substantially protected from drug toxicity. Appearance of tumor was delayed to a median of 28 days in comparison to 12 days for PBS administered controls. We conclude that expression of drug-resistant DHFR in hematopoietic cells confers substantial resistance to MTX, which can be used for more effective chemotherapy against L1210 leukemia in mice. These results support the application of DHFR gene transfer as strategy for improved cancer chemotherapy using MTX and other antifolates.

SPECIFIC AMPLIFICATION OF ILEAL SYMBIONT INTRACELLULARIS FROM SEVERAL ANIMAL SPECIES WITH PROLIFERATIVE ENTERITIS

Proliferative enteritis (PE) is an enteric disease that occurs at weanling age in a variety of animals, including the pig, hamster and horse.4,6,18 There are a variety of clinical presentations, including subclinical disease, reduced weight gain, diarrhoea, and an acute hemorrhagic form. Lesions of PE also vary, but all cases present with hyperplasia of the ileal crypt epithelium and large numbers of small, curved intracellular bacteria within the apical cytoplasm of crypt enterocytes (Fig. 1 and 2). These bacteria can be demonstrated with silver staining18 Other lesions such as gross thickening of the mucosa, inflammation, necrosis, and hemorrhage are variable and may represent complications with concurrent infections, variable chronicity, or host factors.18 The intracellular bacterium of PE has been referred to as a Campylobacter-like organism because of its morphology. However, 16S rDNA sequencing of the organism purified from pig and hamster intestines has shown it to share approximately 90% homology with Desulfovibrio desulfuricans and it has been given the vernacular name Ileal Symbiont (IS) intracellularis.7,17 Transmission studies in hamsters and pigs have confirmed the role of IS intracellularis as the causative agent of PE13, 16, 19.