Summer 2012 Testing and Analysis of the Chemical Mixture Methodology -- Part I

This report presents the key findings made by the Chemical Mixture Methodology (CMM) project team during the first stage of their summer 2012 testing and analysis of the CMM. The study focused on answering the following questions: o What is the percentage of the chemicals in the CMM Rev 27 database associated with each Health Code Number (HCN)? How does this result influence the relative importance of acute HCNs and chronic HCNs in the CMM data set? o What is the benefit of using the HCN-based approach? Which Modes of Action and Target Organ Effects tend to be important in determining the HCN-based Hazard Index (HI) for a chemical mixture? o What are some of the potential issues associated with the current HCN-based approach? What are the opportunities for improving the performance and/or technical defensibility of the HCN-based approach? How would those improvements increase the benefit of using the HCN-based approach? o What is the Target Organ System Effect approach and how can it be used to improve upon the current HCN-based approach? How does the benefits users would derive from using the Target Organ System Approach compare to the benefits available from the current HCN-based approach

On the Action of Methotrexate and 6-Mercaptopurine on M. avium Subspecies paratuberculosis

BACKGROUND: Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP. METHODOLOGY: The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and M. avium using a radiometric ((14)CO(2) BACTEC®) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (% −ΔcGI). PRINCIPAL FINDINGS: The positive control antibiotic (clarithromycin) has ≥85% −ΔcGI at a concentration of 0.5 µg/ml. The negative control (ampicillin) has minimal inhibition at 64 µg/ml. MAP ATCC 19698 shows ≥80% −ΔcGI for both agents by 4 µg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate. CONCLUSIONS: We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % −ΔcGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups

Cancer immunotherapy by immunosuppression

We have previously suggested that the stimulatory effect of a weak immune reaction on tumor growth may be necessary for the growth of incipient tumors. In the present paper, we enlarge upon and extend that idea by collecting evidence in the literature bearing upon this new hypothesis that a growing cancer, whether in man or mouse, is throughout its lifespan, probably growing and progressing because of continued immune stimulation by a weak immune reaction. We also suggest that prolonged immunosuppression might interfere with progression and thus be an aid to therapy. While most of the considerable evidence that supports the hypothesis comes from observations of experimental mouse tumors, there is suggestive evidence that human tumors may behave in much the same way, and as far as we can ascertain, there is no present evidence that necessarily refutes the hypothesis

The Combined Influence of Oral Contraceptives and Human Papillomavirus Virus on Cutaneous Squamous Cell Carcinoma

The vast majority of cutaneous squamous cell carcinoma (CSCC) will occur in those with fair complexion, tendency to burn, and high ultraviolet radiation (UVR) exposure. Organ transplant recipients also are an important population at great risk for CSCC. An association has been reported between oral contraceptive (OC) use, human papillomavirus virus (HPV) and cervical cancer, and there could be a similar association for CSCC. The cutaneous HPV β-E6 protein, a close cousin of the transformative E6 protein underlying anogenital cancers, has been shown to inhibit apoptosis in response to UVR damage and stimulate morphologic transformation in rodent fibroblast cell lines. Furthermore, OC use has been shown to enhance HPV transcription and may contribute to CSCC risk through this pathway

Bicistronic Lentiviruses Containing a Viral 2A Cleavage Sequence Reliably Co-Express Two Proteins and Restore Vision to an Animal Model of LCA1

The disease processes underlying inherited retinal disease are complex and are not completely understood. Many of the corrective gene therapies designed to treat diseases linked to mutations in genes specifically expressed in photoreceptor cells restore function to these cells but fail to stop progression of the disease. There is growing consensus that effective treatments for these diseases will require delivery of multiple therapeutic proteins that will be selected to treat specific aspects of the disease process. The purpose of this study was to design a lentiviral transgene that reliably expresses all of the proteins it encodes and does so in a consistent manner among infected cells. We show, using both in vitro and in vivo analyses, that bicistronic lentiviral transgenes encoding two fluorescent proteins fused to a viral 2A-like cleavage peptide meet these expression criteria. To determine if this transgene design is suitable for therapeutic applications, we replaced one of the fluorescent protein genes with the gene encoding guanylate cyclase -1 (GC1) and delivered lentivirus carrying this transgene to the retinas of the GUCY1*B avian model of Leber congenital amaurosis – 1 (LCA1). GUCY1*B chickens carry a null mutation in the GC1 gene that disrupts photoreceptor function and causes blindness at hatching, a phenotype that closely matches that observed in humans with LCA1. We found that treatment of these animals with the 2A lentivector encoding GC1 restored vision to these animals as evidenced by the presence of optokinetic reflexes. We conclude that 2A-like peptides, with proper optimization, can be successfully incorporated into therapeutic vectors designed to deliver multiple proteins to neural retinal. These results highlight the potential of this vector design to serve as a platform for the development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies