Chimpanzee: A predictive model for the human cytochrome P450 3A subfamily

The human cytochrome P450 3A (CYP3A) subfamily is responsible for most of the metabolism of therapeutic drugs; however, an adequate in vivo model has yet to be discovered. This study begins with an investigation of a controversial topic surrounding the human CYP3As--estrogen regulation. A novel approach to this topic was used by defining expression in the estrogen-responsive endometrium. This study shows that estrogen down-regulates CYP3A4 expression in the endometrium. On the other hand, analogous studies showed an increase in CYP3A expression as age increases in liver tissue. Following the discussion of estrogen regulation, is an investigation of the cross-species relationships among all of the CYP3As was completed. The study compares isoforms from piscines, avians, rodents, canines, ovines, bovines, and primates. Using the traditional phylogenetic analyses and employing a novel approach using exon and intron lengths, the results show that only another primate could be the best animal model for analysis of the regulation of the expression of the human CYP3As. This analysis also demonstrated that the chimpanzee seems to be the best available human model. Moreover, the study showed the presence and similarities of one additional isoform in the chimpanzee genome that is absent in humans. Based on these results, initial characterization of the chimpanzee CYP3A subfamily was begun. While the human genome contains four isoforms--CYP3A4, CYP3A5, CYP3A7, and CYP3A43--the chimpanzee genome has five, the four previously mentioned and CYP3A67. Both species express CYP3A4, CYP3A5, and CYP3A43, but humans express CYP3A7 while chimpanzees express CYP3A67. In humans, CYP3A4 is expressed at higher levels than the other isoforms, but some chimpanzee individuals express CYP3A67 at higher levels than CYP3A4. Such a difference is expected to alter significantly the total CYP3A metabolism. On the other hand, any study considering individual isoforms would still constitute a valid method of study for the human CYP3A4, CYP3A5, and CYP3A43 isoforms. ^

EFFICACY AND MECHANISM OF â-DEFENSIN2 FUSED ANTIGEN PROTEIN VACCINES

Vaccines which use the strategy of fusing adjuvant murine â-defensin2 (mBD2) to an antigen in order to elicit stronger anti-antigen immune responses are referred to as murine â-defensin2 (mBD2) vaccines. Previous studies have validated the potential of mBD2 vaccines, thus in this study we focus on increasing vaccine efficacy as well as mechanism elucidation. Initially, we demonstrate superior IFN-ã release levels by antigen specific effector T cells when antigen is crosspresented by dendritic cells (DC) which absorbed mBD2 vaccine (mBD2 fused antigen protein) over antigen alone. We move unto an in vivo model and note significant increases in the expansion of antigen specific class I T cells but not class II T cells when receiving mBD2 vaccine over antigen alone. Further, knowing mBD2’s link with CC chemokine receptor 6 (CCR6) and Toll-like receptor 4 (TLR4) we note that this enhanced class I T cell expansion is CCR6 independent but TLR4 dependent. With anti-tumor responses desired, we demonstrate in tumor protection experiments with mice, compelling tumor protection when combining adoptive T cell therapy and mBD2 vaccine immunization. We further note that mBD2 vaccines are not limited by the antigen and characterize a viable strategy for enhancing tumor antigen immunogenicity.

Effects of gossypol on DNA replication in mammalian cells

Gossypol, a binaphthalene compound, possesses male infertility effects. However, its mechanism of action and effects on somatic cells are not yet understood. The purpose of this study was to examine the effects of gossypol on mammalian cell growth and DNA replication, using tissue culture cells (HeLa) as an in vivo model.^ Gossypol inhibited DNA synthesis in HeLa cells at low doses, without affecting RNA or protein synthesis. This caused cells to accumulate in S phase without affecting cells in other phases of the cell cycle. The inhibition of DNA synthesis was both dose- and time-dependent. This irreversible block was associated with a decrease in HeLa plating efficiency. Gossypol did bind to DNA but did not measurably affect its ability to serve as a template for DNA polymerase $\alpha$, the major replicative enzyme. Only in the absence of serum could gossypol induce single-strand DNA breaks in HeLa cells; no DNA-DNA or DNA-protein crosslinks were formed.^ Gossypol exhibited dose-dependent inhibition of a number of eukaryotic and prokaryotic replicative DNA polymerases both in vitro and in vivo. This inhibition was kinetically non-competitive with respect to the DNA template and dNTP substrates. Both a filter binding assay and polyacrylamide gel electrophoresis were used to study gossypol binding to DNA polymerase. Inhibition resulted from drug binding to two adjacent amino acid residues on the enzyme. Binding was found to be irreversible and mediated through either non-covalent interactions or by Schiff's base formation between the aldehyde groups of gossypol and the $\varepsilon$-NH$\sb2$ groups of amino acid residues on the polymerase. Structure-function studies using eleven gossypol derivatives revealed that both aldehyde and hydroxyl groups function independently to effect inhibition of DNA polymerase and DNA replication. The activities of DNA polymerase $\beta$ and ribonucleotide reductase were also inhibited by increasing gossypol concentrations.^ These studies demonstrate that the gossypol-mediated inhibition of DNA replication is due in part to inhibition of key replicative enzymes, such as DNA polymerase $\alpha$. The study of DNA polymerase may serve as a model for the interaction of enzymes with gossypol, a drug which may prove useful as a chemotherapeutic agent. ^

Enhanced in vivo angiogenesis within a model tissue engineered construct using biodegradable microspheres containing encapsulated VEGF

Introduction. Tissue engineering techniques offer a potential means to develop a tissue engineered construct (TEC) for the treatment of tissue and organ deficiencies. However, a lack of adequate vascularization is a limiting factor in the development of most viable engineered tissues. Vascular endothelial growth factor (VEGF) could aid in the development of a viable vascular network within TECs. The long-term goals of this research are to develop clinically relevant, appropriately vascularized TECs for use in humans. This project tested the hypothesis that the delivery of VEGF via controlled release from biodegradable microspheres would increase the vascular density and rate of angiogenesis within a model TEC. ^ Materials and methods. Biodegradable VEGF-encapsulated microspheres were manufactured using a novel method entitled the Solid Encapsulation/Single Emulsion/Solvent Extraction technique. Using a PLGA/PEG polymer blend, microspheres were manufactured and characterized in vitro. A model TEC using fibrin was designed for in vivo tissue engineering experimentation. At the appropriate timepoint, the TECs were explanted, and stained and quantified for CD31 using a novel semi-automated thresholding technique. ^ Results. In vitro results show the microspheres could be manufactured, stored, degrade, and release biologically active VEGF. The in vivo investigations revealed that skeletal muscle was the optimal implantation site as compared to dermis. In addition, the TECs containing fibrin with VEGF demonstrated significantly more angiogenesis than the controls. The TECs containing VEGF microspheres displayed a significant increase in vascular density by day 10. Furthermore, TECs containing VEGF microspheres had a significantly increased relative rate of angiogenesis from implantation day 5 to day 10. ^ Conclusions. A novel technique for producing microspheres loaded with biologically active proteins was developed. A defined concentration of microspheres can deliver a quantifiable level of VEGF with known release kinetics. A novel model TEC for in vivo tissue engineering investigations was developed. VEGF and VEGF microspheres stimulate angiogenesis within the model TEC. This investigation determined that biodegradable rhVEGF 165-encapsulated microspheres increased the vascular density and relative rate of angiogenesis within a model TEC. Future applications could include the incorporation of microvascular fragments into the model TEC and the incorporation of specific tissues, such as fat or bone. ^

Molecular basis of Corynebacterium diphtheriae virulence and infection in the Caenorhabditis elegans model host

Corynebacterium diphtheriae is the causative agent of cutaneous and pharyngeal diphtheria in humans. While lethality is certainly caused by diphtheria toxin, corynebacterial colonization may primarily require proteinaceous fibers called pili, which mediate adherence to specific tissues. The type strain of C. diphtheriae possesses three distinct pilus structures, namely the SpaA, SpaD, and SpaH-type pili, which are encoded by three distinct pilus gene clusters. The pilus is assembled onto the bacterial peptidoglycan by a specific transpeptidase enzyme called sortase. Although the SpaA pili are shown to be specific for pharyngeal cells in vitro, little is known about functions of the three pili in bacterial pathogenesis. This is mainly due to lack of in vivo models of corynebacterial infection. As an alternative to mouse models as mice do not have functional receptors for diphtheria toxin, in this study I use Caenorhabditis elegans as a model host for C. diphtheriae. A simple C. elegans model would be beneficial in determining the specific role of each pilus-type and the literature suggests that C. elegans infection model can be used to study a variety of bacterial species giving insight into bacterial virulence and host-pathogen interactions. My study examines the hypothesis that pili and toxin are major virulent determinants of C. diphtheriae in the C. elegans model host.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

A biophysical model of synaptic plasticity and metaplasticity can account for the dynamics of the backward shift of hippocampal place fields.

Hippocampal place cells in the rat undergo experience-dependent changes when the rat runs stereotyped routes. One such change, the backward shift of the place field center of mass, has been linked by previous modeling efforts to spike-timing-dependent plasticity (STDP). However, these models did not account for the termination of the place field shift and they were based on an abstract implementation of STDP that ignores many of the features found in cortical plasticity. Here, instead of the abstract STDP model, we use a calcium-dependent plasticity (CaDP) learning rule that can account for many of the observed properties of cortical plasticity. We use the CaDP learning rule in combination with a model of metaplasticity to simulate place field dynamics. Without any major changes to the parameters of the original model, the present simulations account both for the initial rapid place field shift and for the subsequent slowing down of this shift. These results suggest that the CaDP model captures the essence of a general cortical mechanism of synaptic plasticity, which may underlie numerous forms of synaptic plasticity observed both in vivo and in vitro.

IN-VIVO CT DOSIMETRY DURING VIRTUAL COLONOSCOPY

Virtual colonoscopy (VC) is a minimally invasive means for identifying colorectal polyps and colorectal lesions by insufflating a patient’s bowel, applying contrast agent via rectal catheter, and performing multi-detector computed tomography (MDCT) scans. The technique is recommended for colonic health screening by the American Cancer Society but not funded by the Centers for Medicare and Medicaid Services (CMS) partially because of potential risks from radiation exposure. To date, no in‐vivo organ dose measurements have been performed for MDCT scans; thus, the accuracy of any current dose estimates is currently unknown. In this study, two TLDs were affixed to the inner lumen of standard rectal catheters used in VC, and in-vivo rectal dose measurements were obtained within 6 VC patients. In order to calculate rectal dose, TLD-100 powder response was characterized at diagnostic doses such that appropriate correction factors could be determined for VC. A third-order polynomial regression with a goodness of fit factor of R2=0.992 was constructed from this data. Rectal dose measurements were acquired with TLDs during simulated VC within a modified anthropomorphic phantom configured to represent three sizes of patients undergoing VC. The measured rectal doses decreased in an exponential manner with increasing phantom effective diameter, with R2=0.993 for the exponential regression model and a maximum percent coefficient of variation (%CoV) of 4.33%. In-vivo measurements yielded rectal doses ranged from that decreased exponentially with increasing patient effective diameter, in a manner that was also favorably predicted by the size specific dose estimate (SSDE) model for all VC patients that were of similar age, body composition, and TLD placement. The measured rectal dose within a younger patient was favorably predicted by the anthropomorphic phantom dose regression model due to similarities in the percentages of highly attenuating material at the respective measurement locations and in the placement of the TLDs. The in-vivo TLD response did not increase in %CoV with decreasing dose, and the largest %CoV was 10.0%.

Quantitative MRI of spinal cord injury in a rat model: Correlative studies

Serial quantitative and correlative studies of experimental spinal cord injury (SCI) in rats were conducted using three-dimensional magnetic resonance imaging (MRI). Correlative measures included morphological histopathology, neurobehavioral measures of functional deficit, and biochemical assays for N-acetyl-aspartate (NAA), lactate, pyruvate, and ATP. A spinal cord injury device was characterized and provided a reproducible injury severity. Injuries were moderate and consistent to within $\pm$20% (standard deviation). For MRI, a three-dimensional implementation of the single spin-echo FATE (Fast optimum angle, short TE) pulse sequence was used for rapid acquisition, with a 128 x 128 x 32 (x,y,z) matrix size and a 0.21 x 0.21 x 1.5 mm resolution. These serial studies revealed a bimodal characteristic in the evolution in MRI pathology with time. Early and late phases of SCI pathology were clearly visualized in $T\sb2$-weighted MRI, and these corresponded to specific histopathological changes in the spinal cord. Centralized hypointense MRI regions correlated with evidence of hemorrhagic and necrotic tissue, while surrounding hyperintense regions represented edema or myelomalacia. Unexpectedly, $T\sb2$-weighted MRI pathology contrast at 24 hours after injury appeared to subside before peaking at 72 hours after injury. This change is likely attributable to ongoing secondary injury processes, which may alter local $T\sb2$ values or reduce the natural anisotropy of the spinal cord. MRI, functional, and histological measures all indicated that 72 hours after injury was the temporal maximum for quantitative measures of spinal cord pathology. Thereafter, significant improvement was seen only in neurobehavioral scores. Significant correlations were found between quantitated MRI pathology and histopathology. Also, NAA and lactate levels correlated with behavioral measures of the level of function deficit. Asymmetric (rostral/caudal) changes in NAA and lactate due to injury indicate that rostral and caudal segments from the injury site are affected differently by the injury. These studies indicate that volumetric quantitation of MRI pathology from $T\sb2$-weighted images may play an important role in early prediction of neurologic deficit and spinal cord pathology. The loss of $T\sb2$ contrast at 24 hours suggests MR may be able to detect certain delayed mechanisms of secondary injury which are not resolved by histopathology or other radiological modalities. Furthermore, in vivo proton magnetic resonance spectroscopy (MRS) studies of SCI may provide a valuable addition source of information about changes in regional spinal cord lactate and NAA levels, which are indicative of local metabolic and pathological changes. ^

{\it In vivo\/} induction of cytotoxic T lymphocytes against human immunodeficiency virus type 1 by synthetic viral peptides

Cytotoxic T lymphocytes (CTLs) play an important role in the suppression of initial viremia after acute infection with the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). Most HIV-infected individuals attain a high titer of anti-HIV antibodies within weeks of infection; however this antibody-mediated immune response appears not to be protective. In addition, anti-HIV antibodies can be detrimental to the immune response to HIV through enhancement of infection and participating in autoimmune reactions as a result of HIV protein mimicry of self antigens. Thus induction and maintenance of a strong HIV-specific CTL immune response in the absence of anti-HIV antibodies has been proposed to be the most effective means of controlling of HIV infection. Immunization with synthetic peptides representing HIV-specific CTL epitopes provides a way to induce specific CTL responses, while avoiding stimulation of anti-HIV antibody. This dissertation examines the capacity of synthetic peptides from the V3 loop region of the gp120 envelope protein from several different strain of HIV-1 to induce HIV-specific, MHC-restricted CD8$\sp+$ CTL response in vivo in a mouse model. Seven synthetic peptides representative of sequences found throughout North America, Europe, and Central Africa have been shown to prime CTLs in vivo. In the case of the MN strain of HIV-1, a 13 amino acid sequence defining the epitope is most efficient for optimal induction of specific CTL, whereas eight to nine amino acid sequences that could define the epitope were not immunogenic. In addition, synthesis of peptides with specific amino acid substitutions that are important for either MHC binding or T cell receptor recognition resulted in peptides that exhibited increased immunogenicity and induced CTLs that displayed altered specificity. V3 loop peptides from HIV-1 MN, SC, and Z321 induced a CTL population that was broadly cross-reactive against strains of HIV-1 found throughout the world. This research confirms the potential efficacy of using synthetic peptides for in vivo immunization to induce HIV-specific CTL-mediated responses and provides a basis for further research into development of synthetic peptide-based vaccines. ^

Neuromuscular disuse and atrophy: {\it In vivo\/} magnetic resonance studies of intramuscular lipid metabolism and fluid shift changes

We postulated that neuromuscular disuse results in deleteriously affected tissue-vascular fluid exchange processes and subsequently damages the important oxidative bioenergetic process of intramuscular lipid metabolism. The in-depth research reported in the literature is somewhat limited by the ex vivo nature and sporadic time-course characterization of disuse atrophy and recovery. Thus, an in vivo controlled, localized animal model of disuse atrophy was developed in one of the hindlimbs of laboratory rabbits (employing surgically implanted tetrodotoxin (TTX)-filled mini-osmotic pump-sciatic nerve superfusion system) and tested repeatedly with magnetic resonance (MR) throughout the 2-week period of temporarily induced disuse and during the recovery period (following explantation of the TTX-filled pump) for a period of 3 weeks. Controls consisted of saline/"sham"-implanted rabbit hindlimbs. The validity of this model was established with repeated electrophysiologic nerve conduction testing using a clinically appropriate protocol and percutaneously inserted small needle stimulating and recording electrodes. Evoked responses recorded from proximal (P) and distal (D) sites to the sciatic nerve cuff in the TTX-implanted group revealed significantly decreased (p $<$ 0.001) proximal-to-distal (P/D) amplitude ratios (as much as 50-70% below Baseline/pre-implanted and sham-implanted group values) and significantly increased (p $<$ 0.01) differential latency (PL-DL) values (as much as 1.5 times the pre- and sham-implanted groups). By Day 21 of recovery, observed P/D and PL-DL levels matched Baseline/sham-implemented levels. MRI-determined cross-sectional area (CSA) values of Baseline/pre-implanted, sham- or TTX-implanted, and recovering/explanted and the corresponding contralateral hindlimb tibialis anterior (TA) muscles normalized to tibial bone (TB) CSA (in TA/TB ratios) revealed that there was a significant decline (indicative of atrophic response) from pre- and sham-implanted controls by as much as 20% (p $<$ 0.01) at Day 7 and 50-55% (p $<$ 0.001) at Day 13 of TTX-implantation. In the non-implanted contralaterals, a significant increase (indicative of hypertrophic response) by as much as 10% (p $<$ 0.025) at Day 7 and 27% (p $<$ 0.001) at Day 13 + TTX was found. The induced atrophic/hypertrophic TA muscles were observed to be fully recovered by Day 21 post-explantation as evidenced by image TA/TB ratios. End-point biopsy results from a small group of rabbits revealed comprehensive atrophy of both Type I and Type II fibers, although the heterogeneity of the response supports the use of image-guided, volume-localized proton magnetic resonance spectroscopy (MRS) to noninvasively assess tissue-level metabolic changes. MRS-determined results of a 0.25cc volume of tissue within implanted limb TA muscles under resting/pre-ischemic, ischemic-stressed, and post-ischemic conditions at timepoints during and following disuse atrophy/recovery revealed significantly increased intramuscular spectral lipid levels, as much as 2-3 times (p $<$ 0.01) the Baseline/pre-implanted values at Day 7 and 6-7 times (p $<$ 0.001) at Day 13 + TTX, which approached normal levels (compared to pre- and sham-implanted groups) by Day 21 of post-explanation recovery. (Abstract shortened by UMI.) ^

Diffusion tensor imaging of rat spinal cord in vivo

Magnetic resonance imaging, with its exquisite soft tissue contrast, is an ideal modality for investigating spinal cord pathology. While conventional MRI techniques are very sensitive for spinal cord pathology, their specificity is somewhat limited. Diffusion MRI is an advanced technique which is a very sensitive and specific indicator of the integrity of white matter tracts. Diffusion imaging has been shown to detect early ischemic changes in white matter, while conventional imaging demonstrates no change. By acquiring the complete apparent diffusion tensor (ADT), tissue diffusion properties can be expressed in terms of quantitative and rotationally invariant parameters. ^ Systematic study of SCI in vivo requires controlled animal models such as the popular rat model. To date, studies of spinal cord using ADT imaging have been performed exclusively in fixed, excised spinal cords, introducing inevitable artifacts and losing the benefits of MRI's noninvasive nature. In vivo imaging reflects the actual in vivo tissue properties, and allows each animal to be imaged at multiple time points, greatly reducing the number of animals required to achieve statistical significance. Because the spinal cord is very small, the available signal-to-noise ratio (SNR) is very low. Prior spin-echo based ADT studies of rat spinal cord have relied on high magnetic field strengths and long imaging times—on the order of 10 hours—for adequate SNR. Such long imaging times are incompatible with in vivo imaging, and are not relevant for imaging the early phases following SCI. Echo planar imaging (EPI) is one of the fastest imaging methods, and is popular for diffusion imaging. However, EPI further lowers the image SNR, and is very sensitive to small imperfections in the magnetic field, such as those introduced by the bony spine. Additionally, The small field-of-view (FOV) needed for spinal cord imaging requires large imaging gradients which generate EPI artifacts. The addition of diffusion gradients introduces yet further artifacts. ^ This work develops a method for rapid EPI-based in vivo diffusion imaging of rat spinal cord. The method involves improving the SNR using an implantable coil; reducing magnetic field inhomogeneities by means of an autoshim, and correcting EPI artifacts by post-processing. New EPI artifacts due to diffusion gradients described, and post-processing correction techniques are developed. ^ These techniques were used to obtain rotationally invariant diffusion parameters from 9 animals in vivo, and were validated using the gold-standard, but slow, spinecho based diffusion sequence. These are the first reported measurements of the ADT in spinal cord in vivo . ^ Many of the techniques described are equally applicable toward imaging of human spinal cord. We anticipate that these techniques will aid in evaluating and optimizing potential therapies, and will lead to improved patient care. ^

Assessment of cone beam computed tomography techniques for imaging lung damage in mice in vivo

Lung damage is a common side effect of chemotherapeutic drugs such as bleomycin. This study used a bleomycin mouse model which simulates the lung damage observed in humans. Noninvasive, in vivo cone-beam computed tomography (CBCT) was used to visualize and quantify fibrotic and inflammatory damage over the entire lung volume of mice. Bleomycin was used to induce pulmonary damage in vivo and the results from two CBCT systems, a micro-CT and flat panel CT (fpCT), were compared to histologic measurements, the standard method of murine lung damage quantification. Twenty C57BL/6 mice were given either 3 U/kg of bleomycin or saline intratracheally. The mice were scanned at baseline, before the administration of bleomycin, and then 10, 14, and 21 days afterward. At each time point, a subset of mice was sacrificed for histologic analysis. The resulting CT images were used to assess lung volume. Percent lung damage (PLD) was calculated for each mouse on both the fpCT (PLDfpcT) and the micro-CT (PLDμCT). Histologic PLD (PLDH) was calculated for each histologic section at each time point (day 10, n = 4; day 14, n = 4; day 21, n = 5; control group, n = 5). A linear regression was applied to the PLDfpCT vs. PLDH, PLDμCT vs. PLDH and PLDfpCT vs. PLDμCT distributions. This study did not demonstrate strong correlations between PLDCT and PLDH. The coefficient of determination, R, was 0.68 for PLDμCT vs. PLDH and 0.75 for the PLD fpCT vs. PLDH. The experimental issues identified from this study were: (1) inconsistent inflation of the lungs from scan to scan, (2) variable distribution of damage (one histologic section not representative of overall lung damage), (3) control mice not scanned with each group of bleomycin mice, (4) two CT systems caused long anesthesia time for the mice, and (5) respiratory gating did not hold the volume of lung constant throughout the scan. Addressing these issues might allow for further improvement of the correlation between PLDCT and PLDH. ^

The in vivo and in vitro formation of sticky DNA and the GAA.TTC trinucleotide repeat associated with Friedreich's ataxia

Friedreich's ataxia is caused by the expansion of the GAA•TTC trinucleotide repeat sequence located in intron 1 of the frataxin gene. The long GAA•TTC repeats are known to form several non-B DNA structures including hairpins, triplexes, parallel DNA and sticky DNA. Therefore it is believed that alternative DNA structures play a role in the loss of mRNA transcript and functional frataxin protein in FRDA patients. We wanted to further elucidate the characteristics for formation and stability of sticky DNA by evaluating the structure in a plasmid based system in vitro and in vivo in Escherichia coli. The negative supercoil density of plasmids harboring different lengths of GAA•TTC repeats, as well as either one or two repeat tracts were studied in E. coli to determine if plasmids containing two long tracts (≥60 repeats) in a direct repeat orientation would have a different topological effect in vivo compared to plasmids that harbored only one GAA•TTC tract or two tracts of < 60 repeats. The experiments revealed that, in fact, sticky DNA forming plasmids had a lower average negative supercoil density (-σ) compared to all other control plasmids used that had the potential to form other non-B DNA structures such as triplexes or Z-DNA. Also, the requirements for in vitro dissociation and reconstitution of the DNA•DNA associated region of sticky DNA were evaluated. Results conclude that the two repeat tracts associate in the presence of negative supercoiling and MgCl 2 or MnCl2 in a time and concentration-dependent manner. Interaction of the repeat sequences was not observed in the absence of negative supercoiling and/or MgCl2 or in the presence of other monovalent or divalent cations, indicating that supercoiling and quite specific cations are needed for the association of sticky DNA. These are the first experiments studying a more specific role of supercoiling and cation influence on this DNA conformation. To support our model of the topological effects of sticky DNA in plasmids, changes in sticky DNA band migration was measured with reference to the linear DNA after treatment with increasing concentrations of ethidium bromide (EtBr). The presence of independent negative supercoil domains was confirmed by this method and found to be segregated by the DNA-DNA associated region. Sequence-specific polyamide molecules were used to test the effect of binding of the ligands to the GAA•TTC repeats on the inhibition of sticky DNA. The destabilization of the sticky DNA conformation in vitro through this binding of the polyamides demonstrated the first conceptual therapeutic approach for the treatment of FRDA at the DNA molecular level. ^ Thus, examining the properties of sticky DNA formed by these long repeat tracts is important in the elucidation of the possible role of sticky DNA in Friedreich's ataxia. ^