Effects of dexamethasone on mRNA abundance of nuclear receptors and hepatic nuclear receptor target genes in neonatal calves

Hepatic nuclear receptors (NR), particularly constitutive androstane receptor (CAR) and pregnane X receptor (PXR), are involved in the coordinated transcriptional control of genes that encode proteins involved in the metabolism and detoxification of xeno- and endobiotics. A broad spectrum of metabolic processes are mediated by NR acting in concert with ligands such as glucocorticoids. This study examined the role of dexamethasone on hepatic mRNA expression of CAR, PXR and several NR target genes. Twenty-eight male calves were allotted to one of four treatment groups in a 2 x 2 arrangement of treatments: feed source (colostrum or milk-based formula) and glucocorticoid administration (twice daily intramuscular dexamethasone). Liver biopsies were obtained at 5 days of age. Real-time reverse transcription polymerase chain reaction was used to quantify mRNA abundances. No effects of feed source on mRNA abundances were observed. For the NR examined, mRNA abundance of both CAR and PXR in dexamethasone-treated calves was lower (p < 0.05) by 39% and 40%, respectively, than in control calves. Abundance of NR target genes exhibited a mixed response. SULT1A1 mRNA abundance was 39% higher (p < 0.05) in dexamethasone-treated calves compared with control calves. mRNA abundance of CYP2C8 tended also to be higher (+44%; p = 0.053) after dexamethasone treatment. No significant treatment effects (p > 0.10) were observed for mRNA abundances of CYP3A4, CYP2E1, SULT2A1, UGT1A1 or cytochrome P450 reductase (CPR). In conclusion, an enhanced glucocorticoid status, induced by pharmacological amounts of dexamethasone, had differential and in part unexpected effects on NR and NR target systems in 5-day-old calves. Part of the unexpected responses may be due the immaturity of NR and NR receptor target systems.

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells.

Bovine colostrogenesis is distinguished by the specific transfer of IgG1 from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E2 ) and progesterone (P4 ) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta-2-Microglobulin (β2M). We hypothesized that steroid hormone treatments (E2 and P4 ) of bovine mammary epithelial cells in vitro would induce up-regulation of IgG1 transcytosis candidate gene mRNA expression suggesting involvement in IgG1 transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up-regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG1 transcytosis in bovine mammary cells.

Effects of induced energy deficiency on lactoferrin concentration in milk and the lactoferrin reaction of primary bovine mammary epithelial cells in vitro.

A dietary energy restriction to 49% of total energy requirements was conducted with Red Holstein cows for three weeks in mid-lactation. At the last day of the restriction phase, primary bovine mammary epithelial cells (pbMEC) of eight restriction (RF) and seven control-fed (CF) cows were extracted out of one litre of milk and cultured. In their third passage, an immune challenge with the most prevalent, heat-inactivated mastitis pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was conducted. Lactoferrin (LF) was determined on gene expression and protein level. An enzyme-linked immunosorbent assay (ELISA) was developed to determine LF in milk samples taken twice weekly throughout the animal trial, beginning on day 20 pp (post-partum) until day 150 pp, in cell culture total protein and in cell culture supernatant. Milk LF increased throughout the lactation and decreased significantly during the induced energy deficiency in the RF group. At the beginning of realimentation, LF concentration increased immediately in the RF group and reached higher levels than before the induced deficit following the upward trend seen in the CF group. Cell culture data revealed higher levels (up to sevenfold up-regulation in gene expression) and significant higher LF protein concentration in the RF compared to the CF group cells. A further emphasized effect was found in E. coli compared to S. aureus exposed cells. The general elevated LF levels in the RF pbMEC group and the further increase owing to the immune challenge indicate an unexpected memory ability of milk-extracted mammary cells that were transposed into in vitro conditions and even displayed in the third passage of cultivation. The study confirms the suitability of the non-invasive milk-extracted pbMEC culture model to monitor the influence of feeding experiments on immunological situations in vivo.

Inherited and noninherited sources of variation in metastasis and the growth rate of tumors: Implications for tumor evolution and therapy

I have undertaken measurements of the genetic (or inherited) and nongenetic (or noninherited) components of the variability of metastasis formation and tumor diameter doubling time in more than 100 metastatic lines from each of three murine tumors (sarcoma SANH, sarcoma SA4020, and hepatocarcinoma HCA-I) syngeneic to C3Hf/Kam mice. These lines were isolated twice from lung metastases and analysed immediately thereafter to obtain the variance to spontaneous lung metastasis and tumor diameter doubling time. Additional studies utilized cells obtained from within 4 passages of isolation. Under the assumption that no genetic differences in metastasis formation or diameter doubling time existed among the cells of a given line, the variance within a line would estimate nongenetic variation. The variability derived from differences between lines would represent genetic origin. The estimates of the genetic contribution to the variation of metastasis and tumor diameter doubling time were significantly greater than zero, but only in the metastatic lines of tumor SANH was genetic variation the major source of metastatic variability (contributing 53% of the variability). In the tumor cell lines of SA4020 and HCA-I, however, the contribution of nongenetic factors predominated over genetic factors in the variability of the number of metastasis and tumor diameter doubling time. A number of other parameters examined, such as DNA content, karyotype, and selection and variance analysis with passage in vivo, indicated that genetic differences existed within the cell lines and that these differences were probably created by genetic instability. The mean metastatic propensity of the lines may have increased somewhat during their isolation and isotransplantation, but the variance was only slightly affected, if at all. Analysis of the DNA profiles of the metastatic lines of SA4020 and HCA-I revealed differences between these lines and their primary parent tumors, but not among the SANH lines and their parent tumor. Furthermore, there was a direct correlation between the extent of genetic influence on metastasis formation and the ability of the tumor cells to develop resistance to cisplatinum. Thus although nongenetic factors might predominate in contributing to metastasis formation, it is probably genetic variation and genetic instability that cause the progression of tumor cells to a more metastatic phenotype and leads to the emergence of drug resistance. ^

{\it In vivo\/} magnetic resonance studies of experimental liver disease: Carbon tetrachloride hepatotoxicity and alcohol-induced fatty liver in rat

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) were used to non-invasively determine if cirrhosis induced by carbon tetrachloride (CCl$\sb4$) and phospholipase-D (PLD) could be distinguished from fatty infiltration in rat. MRS localization and water suppression methods were developed, implemented and evaluated in terms of their application to in vivo proton NMR studies of experimental liver disease. MRS studies were also performed to quantitate fatty infiltration resulting from carbon tetrachloride (CCl$\sb4$) or alcohol (ethanol) administration and the MRS results were confirmed using biochemical total lipid analysis and histology. $\rm T\sb1$ weighted MR images acquired weekly, 48 hours post administration, demonstrated only a slight increase in overall liver intensity with CCl$\sb4$ or alcohol administration, which is consistent with previously reported results. The MR images were able to detect nodules resulting from CCl$\sb4$+PLD induced cirrhosis as hypointense regions, also consistent with previous reports. Localized in vivo water and lipid proton $\rm T\sb1$ relaxation time measurements were performed and demonstrated no statistically significant trends for either agent. In vivo proton spectra were also acquired using stimulated echo techniques to quantitatively follow the changes in liver lipid content. The changes in liver lipid content observed using MRS were verified by total lipid analysis using the Folch technique and histology. The in vivo $\rm T\sb1$ and lipid quantification data str inconsistent with the previous hypothesis that the changes in $\rm T\sb1$ weighted images were the result of increased "free" water content and, therefore, increased water $\rm T\sb1$ relaxation times. These data indicate that the long term changes are more likely the result of changes in lipid content. The data are also shown to agree with the accepted hypothesis that the time course and mechanism of fatty infiltration are different for CCl$\sb4$ and alcohol. The hypothesis that the lipids resulting from either protocol are from the same lipid fraction(s), presumably triglycerides, is also supported. And lastly, on the basis of MR images and quantitative MRS lipid information, it was shown that cirrhosis could be distinguished from fatty infiltration. ^

A convolution model for energy transport in a therapeutic fast neutron beam

A three-dimensional model has been proposed that uses Monte Carlo and fast Fourier transform convolution techniques to calculate the dose distribution from a fast neutron beam. This method transports scattered neutrons and photons in the forward, lateral, and backward directions and protons, electrons, and positrons in the forward and lateral directions by convolving energy spread kernels with initial interaction available energy distributions. The primary neutron and photon spectrums have been derived from narrow beam attenuation measurements. The positions and strengths of the effective primary neutron, scattered neutron, and photon sources have been derived from dual ion chamber measurements. The size of the effective primary neutron source has been measured using a copper activation technique. Heterogeneous tissue calculations require a weighted sum of two convolutions for each component since the kernels must be invariant for FFT convolution. Comparisons between calculations and measurements were performed for several water and heterogeneous phantom geometries. ^

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. ^

Development of automated image analysis tools for verification of radiotherapy field accuracy with an electronic portal imaging device

The successful management of cancer with radiation relies on the accurate deposition of a prescribed dose to a prescribed anatomical volume within the patient. Treatment set-up errors are inevitable because the alignment of field shaping devices with the patient must be repeated daily up to eighty times during the course of a fractionated radiotherapy treatment. With the invention of electronic portal imaging devices (EPIDs), patient's portal images can be visualized daily in real-time after only a small fraction of the radiation dose has been delivered to each treatment field. However, the accuracy of human visual evaluation of low-contrast portal images has been found to be inadequate. The goal of this research is to develop automated image analysis tools to detect both treatment field shape errors and patient anatomy placement errors with an EPID. A moments method has been developed to align treatment field images to compensate for lack of repositioning precision of the image detector. A figure of merit has also been established to verify the shape and rotation of the treatment fields. Following proper alignment of treatment field boundaries, a cross-correlation method has been developed to detect shifts of the patient's anatomy relative to the treatment field boundary. Phantom studies showed that the moments method aligned the radiation fields to within 0.5mm of translation and 0.5$\sp\circ$ of rotation and that the cross-correlation method aligned anatomical structures inside the radiation field to within 1 mm of translation and 1$\sp\circ$ of rotation. A new procedure of generating and using digitally reconstructed radiographs (DRRs) at megavoltage energies as reference images was also investigated. The procedure allowed a direct comparison between a designed treatment portal and the actual patient setup positions detected by an EPID. Phantom studies confirmed the feasibility of the methodology. Both the moments method and the cross-correlation technique were implemented within an experimental radiotherapy picture archival and communication system (RT-PACS) and were used clinically to evaluate the setup variability of two groups of cancer patients treated with and without an alpha-cradle immobilization aid. The tools developed in this project have proven to be very effective and have played an important role in detecting patient alignment errors and field-shape errors in treatment fields formed by a multileaf collimator (MLC). ^

Automatic quantitation of multiple sclerosis lesions on MR images

A two-pronged approach for the automatic quantitation of multiple sclerosis (MS) lesions on magnetic resonance (MR) images has been developed. This method includes the design and use of a pulse sequence for improved lesion-to-tissue contrast (LTC) and seeks to identify and minimize the sources of false lesion classifications in segmented images. The new pulse sequence, referred to as AFFIRMATIVE (Attenuation of Fluid by Fast Inversion Recovery with MAgnetization Transfer Imaging with Variable Echoes), improves the LTC, relative to spin-echo images, by combining Fluid-Attenuated Inversion Recovery (FLAIR) and Magnetization Transfer Contrast (MTC). In addition to acquiring fast FLAIR/MTC images, the AFFIRMATIVE sequence simultaneously acquires fast spin-echo (FSE) images for spatial registration of images, which is necessary for accurate lesion quantitation. Flow has been found to be a primary source of false lesion classifications. Therefore, an imaging protocol and reconstruction methods are developed to generate "flow images" which depict both coherent (vascular) and incoherent (CSF) flow. An automatic technique is designed for the removal of extra-meningeal tissues, since these are known to be sources of false lesion classifications. A retrospective, three-dimensional (3D) registration algorithm is implemented to correct for patient movement which may have occurred between AFFIRMATIVE and flow imaging scans. Following application of these pre-processing steps, images are segmented into white matter, gray matter, cerebrospinal fluid, and MS lesions based on AFFIRMATIVE and flow images using an automatic algorithm. All algorithms are seamlessly integrated into a single MR image analysis software package. Lesion quantitation has been performed on images from 15 patient volunteers. The total processing time is less than two hours per patient on a SPARCstation 20. The automated nature of this approach should provide an objective means of monitoring the progression, stabilization, and/or regression of MS lesions in large-scale, multi-center clinical trials. ^

Tumors in the contralateral breast following radiation therapy for breast cancer

The proportional distribution of independent malignant tumors in the contralateral breast following treatment for breast cancer was investigated to assess the influence of scattered radiation as a cause of these tumors. In a population of 172 patients the proportion of contralateral tumors in each quadrant and the center (the nipple-areolar complex) was compared with the expected, or natural, distribution found in the general population, in the absence of radiation. The observed/expected ratio for contralateral tumors was 1.43 for the upper-inner quadrant; 0.97, lower-inner quadrant; 1.51, center; 0.76, upper-outer quadrant; and 0.64, lower-outer quadrant. In each quadrant, except the lower-inner, the observed/expected ratio differed from 1.00 with statistical significance at the 5% level (one-tail). The same analysis, stratified by age and menopausal status, showed a similar shift of tumors, with more than expected in the inner quadrants and center and less than expected in the outer quadrants, although the results did not show statistical significance at the 5% level for all strata. For each patient the mean absorbed radiation dose for each quadrant and center of the breast was estimated, based on measurements in a tissue-equivalent phantom. Among patients the doses ranged from 0.5 to 8 Gy; within individuals, doses to the inner quadrants typically were a factor of three times higher than doses to the outer quadrants. The results suggest that radiation may be a risk factor for contralateral breast tumors and warrants further investigation. ^

A genetically engineered, adherent smooth muscle cell lining for left ventricular assist devices

Due to the clinical success of left ventricular assist devices (LVADs) used for short term "bridge to transplant" and the limited availability of donor organs, heart assist devices are being considered for long term implantation as an alternative to heart transplantation. In an effort to improve biocompatibility, a nonthrombogenic cellular lining was developed from genetically engineered smooth muscle cells (GE-SMC) for the Thermocardiosystems Heartmate$\sp{\rm TM}$ LVAD. SMCs have been transduced with the genes for endothelial nitric oxide synthase (NOS III) and GTP cyclohydrolase (GTPCH) with subsequent stable expression of the NOS III protein via an Epstein Barr based DNA expression vector. Transduced SMCs produce nitric oxide at concentrations that reduce platelet deposition and smooth muscle cell proliferation when tested in vitro. In addition, the adhesive capabilities of GE-SMC linings were also examined, and optimized in physical environments mimicking typical in vivo LVAD operation. Preliminary investigations examining cell adhesion during constant shear stress exposure demonstrated an acute phase of cell loss corresponding to cytoskeletal F-actin rearrangement. Subsequently, an in vitro circulatory loop was designed to expose cell lined LVADs to in vivo operating conditions. Cumulative cell loss from cell lined LVADs was less than 10% after 24 hours of flow. Using a protocol for "preconditioning" the cell lining within the mock circulatory loop, the first implantation of an LVAD containing a genetically engineered SMC lining was successfully implemented in a bovine model. Results from this 24 hour study indicate that the flow-conditioned cellular lining remained intact with no evidence of thromboembolization and only minimal changes in coagulation studies. ^

THE CALIBRATION AND IMPLEMENTATION OF CR-39 NUCLEAR TRACK DETECTOR MATERIAL AS A FAST NEUTRON MICRODOSIMETER

A detailed microdosimetric characterization of the M. D. Anderson 42 MeV (p,Be) fast neutron beam was performed using the techniques of microdosimetry and a 1/2 inch diameter Rossi proportional counter. These measurements were performed at 5, 15, and 30 cm depths on the central axis, 3 cm inside, and 3 cm outside the field edge for 10 $\times$ 10 and 20 $\times$ 20 cm field sizes. Spectra were also measured at 5 and 15 cm depth on central axis for a 6 $\times$ 6 cm field size. Continuous slowing down approximation calculations were performed to model the nuclear processes that occur in the fast neutron beam. Irradiation of the CR-39 was performed using a tandem electrostatic accelerator for protons of 10, 6, and 3 MeV and alpha particles of 15, 10, and 7 MeV incident energy on target at angles of incidence from 0 to 85 degrees. The critical angle as well as track etch rate and normal incidence diameter versus linear energy transfer (LET) were obtained from these measurements. The bulk etch rate was also calculated from these measurements. Dose response of the material was studied, and the angular distribution of charged particles created by the fast neutron beam was measured with CR-39. The efficiency of CR-39 was calculated versus that of the Rossi chamber, and an algorithm was devised for derivation of LET spectra from the major and minor axis dimensions of the observed tracks. The CR-39 was irradiated in the same positions as the Rossi chamber, and the derived spectra were compared directly. ^

RELATIONSHIP OF MOUSE IMMUNOGLOBULIN GENES TO INVERTED REPETITIVE DNA SEQUENCES

The purpose of this study was to examine the relationship of immunoglobulin genes, more specifically the C regions, to the inverted repetitive sequences found in the mouse genome. Total mRNA as well as mRNA for light chain kappa was purified from mouse plasmacytoma MOPC 321 cells. Complementary DNA molecules were synthesized from the mRNA templates and hybridized to DNA fractionated on hydroxyapatite columns. This fractionation separates DNA according to the presence of inverted repetitive sequences which will be retained by hydroxyapatite while the remaining fraction will be unbound.^ The results obtained during the course of this investigation suggested the following conclusions. Firstly, it was shown that inverted sequences were not found within the transcribed DNA region. Secondly, inverted sequences are not found within the kappa gene. And finally, it was shown that the inverted sequences may not be representative of all the sequences found in MOPC 321 DNA. ^