Lance B. Augustin

Transcriptional Regulation of Mouse δ-Opioid Receptor Gene

Three major types of opioid receptors, μ (MOR), δ (DOR), and κ (KOR), have been cloned and characterized. Each opioid receptor exhibits a distinct pharmacological profile as well as a distinct pattern of temporal and spatial expression in the brain, suggesting the critical role of transcription regulatory elements and their associated factors. Here, we report the identification of a minimum core promoter, in the 5′-flanking region of the mouse DOR gene, containing an E box and a GC box that are crucial for DOR promoter activity in NS20Y cells, a DOR-expressing mouse neuronal cell line.In vitro protein-DNA binding assays and in vivotransient transfection assays indicated that members of both the upstream stimulatory factor and Sp families of transcription factors bound to and trans-activated the DOR promoter via the E box and GC box, respectively. Furthermore, functional and physical interactions between these factors were critical for the basal as well as maximum promoter activity of the DOR gene. Thus, the distinct developmental emergence and brain regional distribution of the δ opioid receptor appear to be controlled, at least in part, by these two regulatory elements and their associated factors.

Gene repair and transposon-mediated gene therapy

The main strategy of gene therapy has traditionally been focused on gene augmentation. This approach typically involves the introduction of an expression system designed to express a specific protein in the transfected cell. Both the basic and clinical sciences have generated enough information to suggest that gene therapy would eventually alter the fundamental practice of modern medicine. However, despite progress in the field, widespread clinical applications and success have not been achieved. The myriad deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. One, derived primarily from the pioneering work of homologous recombination, is gene repair. Simply stated, the process involves targeting the mutation in situ for gene correction and a return to normal gene function.

Spontaneous circulation of myeloid-lymphoid–initiating cells and SCID-repopulating cells in sickle cell crisis

The only curative therapy for sickle cell disease (SCD) is allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy approaches for autologous HSC transplantation are being developed. Although earlier engraftment is seen when cells from GCSF-mobilized blood are transplanted than when bone marrow is transplanted, administration of GCSF to patients with SCD can cause significant morbidity. We tested whether primitive hematopoietic progenitors are spontaneously mobilized in the blood of patients with SCD during acute crisis (AC-SCD patients). The frequency of myeloid-lymphoid-initiating cells (ML-ICs) and SCID-repopulating cells (SRCs) was significantly higher in blood from AC-SCD patients than in blood from patients with steady-state SCD or from normal donors. The presence of SRCs in peripheral blood was not associated with detection of long-term culture-initiating cells, consistent with the notion that SRCs are more primitive than long-term culture-initiating cells. As ML-ICs and SRCs were both detected in blood of AC-SCD patients only, these assays may both measure primitive progenitors. The frequency of ML-ICs also correlated with increases in stem cell factor, GCSF, and IL-8 levels in AC-SCD compared with steady-state SCD and normal-donor sera. Because significant numbers of ML-ICs and SRCs are mobilized in the blood without exogenous cytokine treatment during acute crisis of SCD, collection of peripheral blood progenitors during crisis may yield a source of autologous HSCs suitable for ex-vivo correction by gene therapy approaches and subsequent transplantation.

Hepatic expression of the SPOT 14 (S14) paralog S14-Related (Mid1 interacting protein) is regulated by dietary carbohydrate

The Spot 14 (S14) gene is rapidly up-regulated by signals that induce lipogenesis such as enhanced glucose metabolism and thyroid hormone administration. Previous studies in S14 null mice show that S14 is required for normal lipogenesis in the lactating mammary gland, but not the liver. We speculated that the lack of a hepatic phenotype was due to the expression of a compensatory gene. We recently reported that this gene is likely an S14 paralog that we named S14-Related (S14-R). S14-R is expressed in the liver, but not in the mammary gland. If S14-R compensates for the absence of S14 in the liver, we hypothesized that, like S14, S14-R expression should be glucose responsive. Here, we report that hepatic S14-R mRNA levels increase with high-carbohydrate feeding in mice or within 2 h of treating cultured hepatocytes with elevated glucose. A potential carbohydrate response element (ChoRE) was identified at position -458 of the S14-R promoter. Deletion of or point mutations within the putative S14-R ChoRE led to 50-95% inhibition of the glucose response. Gel-shift analysis revealed that the glucose-activated transcription complex carbohydrate responsive element-binding protein/Max-like protein X (Mlx) binds to the S14-R ChoRE. Finally, S14-R glucose induction is completely blocked when a dominant-negative form of Mlx is overexpressed in primary hepatocytes. In conclusion, our results indicate that the S14-R gene is a glucose-responsive target of carbohydrate responsive element-binding protein/Mlx and suggest that the S14-R protein is a compensatory factor, at least partially responsible for the normal liver lipogenesis observed in the S14 null mouse.

Safety and immunogenicity of Salmonella typhimurium expressing C-terminal truncated human IL-2 in a murine model

Salmonella enterica serovar Typhimurium preferentially colonizes tumors in vivo and has proven to be an effective biologic vector. The attenuated S. enterica Typhimurium strain χ4550 was engineered to express truncated human interleukin-2 and renamed SalpIL2. Previously, we observed that a single oral administration of SalpIL2 reduced tumor number and volume, while significantly increasing local and systemic natural killer (NK) cell populations in an experimental metastasis model. Here we report that in nontumor-bearing mice, a single oral dose of SalpIL2 resulted in increased splenic cytotoxic T and NK cell populations that returned to control levels by 4 weeks post oral administration. Though SalpIL2 was detected in mouse tissues for up to 10 weeks, no prolonged alterations in peripheral blood serum chemistry or complete blood cell counts were observed. Similarly, comparative histopathological analysis of tissues revealed no significant increase in pyogranulomas in SalpIL2-treated animals with respect to saline controls. In Rag-1 knockout mice, which have severely impaired B and T cell function, SalpIL2 reduced growth of hepatic metastases. Furthermore, SalpIL2 altered expression of several proinflammatory cytokines and chemokines in the serum of mice with pulmonary osteosarcoma metastases. These data further suggest that SalpIL2 is avirulent and induces a cell-mediated antitumor response.

A phase I clinical study to evaluate safety of orally administered, genetically engineered Salmonella enterica serovar Typhimurium for canine osteosarcoma

Abstract We conducted a prospective phase I study to evaluate safety of an orally administered Salmonella encoding IL‐2 (SalpIL2) in combination with amputation and adjuvant doxorubicin for canine appendicular osteosarcoma. Efficacy was assessed as a secondary measure. The first dose of SalpIL2 was administered to 19 dogs on Day 0; amputation was done after 10 days with chemotherapy following 2 weeks later. SalpIL2 was administered concurrent with chemotherapy, for a total of five doses of doxorubicin and six doses of SalpIL2. There were six reportable events prior to chemotherapy, but none appeared due to SalpIL2. Dogs receiving SalpIL2 had significantly longer disease‐free interval (DFI) than a comparison group of dogs treated with doxorubicin alone. Dogs treated using lower doses of SalpIL2 also had longer DFI than dogs treated using the highest SalpIL2 dose. The data indicate that SalpIL2 is safe and well tolerated, which supports additional testing to establish the potential for SalpIL2 as a novel form of adjuvant therapy for dogs with osteosarcoma.

Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli

Programmed death ligand 1 (PD-L1), is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. Single chain variable fragments (scFvs) have several desirable characteristics and are an attractive alternative to monoclonal antibodies for experimental or therapeutic purposes. Three chickens were immunized against murine PD-L1, and mRNA isolated from their spleens was used to generate an immunized immunoglobulin variable region library. Using splice-overlap extension PCR, variable region cDNAs were combined to generate full-length scFvs. M13 phage display of the resulting scFv library identified a functional scFv against PD-L1 (αPD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6×-His tag using immobile metal affinity chromatography. The dissociation constant of αPD-L1 scFv was determined to be 7.11×10(-10)M, and the scFv demonstrated inhibitory biological activity comparable to an antagonistic monoclonal antibody, providing an alternative agent for blocking PD-1/PD-L1 signaling.

Expression of wild-type and mutant human ornithine transcarbamylase genes in Chinese hamster ovary cells and lack of dominant negative effect of R141Q and R40H mutants.

Chinese hamster ovary cultured cells were transformed to continuously express wild-type and two mutant ornithine transcarbamylase genes, R141Q and R40H. In addition, these cells were transfected to transiently express the same genes. The R141Q mutation abolishes the enzymatic activity, and the amount of “mature” protein present in transfected cells is equivalent to the wild type. The R40H mutation causes a reduction of enzymatic activity to approximately 26 to 35% of wild type concomitant with a significant reduction in the amount of protein present. Transfection with wild-type and mutant genes together in various proportions did not reveal dominant negative effects of the two mutations studied. This expression system can be used to examine the deleterious effect of private mutations or lack thereof in families with ornithine transcarbamylase deficiency as well as evaluate the potential dominant negative effects of gene delivery for treatment of ornithine transcarbamylase deficiency.

Antioxidant oils and Salmonella enterica Typhimurium reduce tumor in an experimental model of hepatic metastasis

Fruit seeds high in antioxidants have been shown to have anticancer properties and enhance host protection against microbial infection. Recently we showed that a single oral dose of Salmonella enterica serovar Typhimurium expressing a truncated human interleukin-2 gene (SalpIL2) is avirulent, immunogenic, and reduces hepatic metastases through increased natural killer cell populations in mice. To determine whether antioxidant compounds enhance the antitumor effect seen in SalpIL2-treated animals, we assayed black cumin (BC), black raspberry (BR), and milk thistle (MT) seed oils for the ability to reduce experimental hepatic metastases in mice. In animals without tumor, BC and BR oil diets altered the kinetics of the splenic lymphocyte response to SalpIL2. Consistent with previous reports, BR and BC seed oils demonstrated independent antitumor properties and moderate adjuvant potential with SalpIL2. MT oil, however, inhibited the efficacy of SalpIL2 in our model. Based on these data, we conclude that a diet high in antioxidant oils promoted a more robust immune response to SalpIL2, thus enhancing its antitumor efficacy.

Low dose chemotherapy combined with attenuated Salmonella decreases tumor burden and is less toxic than high dose chemotherapy in an autochthonous murine model of breast cancer

Background. Immunotherapies for cancer treatment have demonstrated substantial promise even though toxicities and development of tumor resistance limit their effectiveness. A combinatorial approach using immunotherapy with other treatment modalities may decrease side effects while maintaining maximal therapeutic effect. We aimed to determine if bacterial immunotherapy in combination with a chemotherapeutic would be efficacious and less toxic than conventional chemotherapy in an established, preclinical, autochthonous tumor model. Methods. BALB‐neuT mice develop autochthonous mammary neoplasms that resemble closely the aggressive Her2‐driven cancer found in human patients. Virulence‐attenuated S. Typhimurium was used for bacterial immunotherapy. Doxorubicin was the chemotherapeutic agent used at the maximum tolerated dose (5 mg/kg) and low dose (1.25 mg/kg). S. Typhimurium was administered intravenously on day 0 and doxorubicin on days 0, 7, and 14. Experiments concluded on day 35. Mammary pad tumors were measured weekly to ascertain efficacy, and mice were weighed weekly to evaluate toxicity. Results. Mice administered maximum tolerated dose doxorubicin (5 mg/kg) demonstrated a 1.4‐fold increase in tumor size by day 35 and showed a nearly 25% weight loss by day 14 revealing severe toxicity. When mice were administered a single dose of S. Typhimurium combined with a low dose of doxorubicin (1.25 mg/kg), tumors increased <3‐fold by day 35, and mice showed only 5% weight loss, indicating no clinically relevant toxicity. Conclusion. Bacterial immunotherapy combined with low dose chemotherapy decreased the tumor burden when compared with low dose chemotherapy alone and was less toxic than maximum tolerated dose chemotherapy in an established, autochthonous murine model of breast cancer.