Weighing the results of differing 'low dose' studies of the mouse prostate by Nagel, Cagen, and Ashby: quantification of experimental power and statistical results.

Differing experimental findings with respect to "low dose" responses in the mouse prostate after in utero exposure have generated considerable controversy. An analysis of such controversies requires a broad strength and weight of the evidence approach. For example, a National Toxicology Program review panel acquired the raw data from nearly 50 studies and then statistically reanalyzed these data in a common and comparable approach. However, the statistical power of the various studies was not calculated and the quantitative p values were not reported in this reanalysis. Such calculations and values address vital strength- and weight-of-the-evidence questions: (1) how sensitive were the various studies to detect changes in prostate weight, particularly the negative replicate studies and (2) what were the p values; were negative studies robust or only marginal in their inability to find an effect? We first examined the statistical power of the studies to detect a positive effect on prostate weight. Preliminary calculations indicated that the two subsequent replicating studies were indeed more sensitive to changes in prostate weight in comparison to the original study, having reasonable power to detect an effect at only 50% of the response reported in the original study. Additional calculations were performed using the raw data available from one negative replicating study and the methods recommended by the statistics subpanel of the original review. This analysis used Dunnett's multiple comparison procedure for groups with p<0.05 to infer statistical significance, employed an analysis-of-covariance model with body weight as a covariate, and addressed litter as a nested random effect. The quantitated p values for this replicated study, comparing the two Bisphenol A treatment groups (2 and 20 microg/kg/day) to the control, were 0.821 and 0.972, respectively. This indicates this study was indeed robust in finding no treatment-related effect. Thus, the weight and strength of the evidence, based on sensitivity and quantitative p value, was that it is highly unlikely for this negative replicating study to have missed a true effect. In the future, we recommend a similar use of statistical power analysis for those designing experimental studies and for those conducting weight-of-the-evidence reviews, and we also recommend the clear quantitation and reporting of p values to support the review's interpretation and conclusions.

Citrate shows specific, dose-dependent lympholytic activity in neoplastic cell lines.

We have demonstrated cell membrane destruction activity by carboxylic acid derivatives (CADs) mainly tri-sodium citrate, in neoplastic cell lines and, to a far lesser extent, in normal human peripheral blood mononuclear cells (hPBMC). Flow cytometric (FACS) analysis was applied to Annexin-V and Propidium Iodide (PI) stained cells to evaluate the degree of the apoptosis induced by citrate in the following cell lines: CCRF-CEM (shortened to CEM), H9, and Jurkat (T-Cells), Raji and WIL2-NS (B-Cells), HL-60 (myeloblasts), K562 (myelocytes) and U937 (monocytes). We also tested normal hPBMC. Before staining with Annexin/PI, manual cell counts were performed on 24- and 48-h-old cell cultures. Cell supernatants were assayed for lactate dehydrogenase (LDH). LDH values in samples correlated with enhanced apoptosis by FACS analysis. For comparison, ascorbate and 2 other CADs including, acetate and lactate were also evaluated for the induction of apoptosis. In addition, the ability of tri-sodium citrate to induce apoptosis in the presence and the absence of several antineoplastic drugs, such as dexamethasone, arsenic trioxide, hydrocortisone, 6-mercaptopurine, and methotrexate were tested on Jurkat cells. FACS, LDH, and cell count values all demonstrated an enhanced degree of apoptotic cell death in Jurkat cells by citrate. In most of our investigated cells, except for the H9 cell line, citrate has induced a greater degree of apoptosis than acetate which induced a greater degree than lactate (see Fig. 1.0). The nature of the cell death by ascorbate appeared to be due to necrosis rather than apoptosis. Pilot studies on normal hPBMC showed that citrate alone or in combination with antineoplastic drugs caused minimal cell death. Thus citrate might be of benefit in some chemotherapy treatments in order to reduce drug toxicity or possibly enhance drug activities in certain neoplasias.

Paramyxovirus infection in caiman lizards (Draecena guianensis).

Three separate epidemics occurred in caiman lizards (Dracaena guianensis) that were imported into the USA from Peru in late 1998 and early 1999. Histologic evaluation of tissues from necropsied lizards demonstrated a proliferative pneumonia. Electron microscopic examination of lung tissue revealed a virus that was consistent with members of the family Paramyxoviridae. Using a rabbit polyclonal antibody against an isolate of ophidian (snake) paramyxovirus, an immunoperoxidase staining technique demonstrated immunoreactivity within pulmonary epithelial cells of 1 lizard. Homogenates of lung, brain, liver, or kidney from affected lizards were placed in flasks containing monolayers of either terrapene heart cells or viper heart cells. Five to 10 days later, syncytial cells formed. When Vero cells were inoculated with supernatant of infected terrapene heart cells, similar syncytial cells developed. Electron microscopic evaluation of infected terrapene heart cells revealed intracytoplasmic inclusions consisting of nucleocapsid strands. Using negative-staining electron microscopy, abundant filamentous nucleocapsid material with a herringbone structure typical of the Paramyxoviridae was observed in culture medium of infected viper heart cells. Seven months following the initial epizootic, blood samples were collected from surviving group 1 lizards, and a hemagglutination inhibition assay was performed to determine presence of specific antibody against the caiman lizard isolate. Of the 17 lizards sampled, 7 had titers of < or =1:20 and 10 had titers of >1:20 and < or =1:80. This report is only the second of a paramyxovirus identified in a lizard and is the first to snow the relationship between histologic and ultrastructural findings and virus isolation.

ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation.

We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.

Isolation and experimental transmission of a reovirus pathogenic in ratsnakes (Elaphe species).

A reovirus was isolated from juvenile Moellendorff's ratsnakes (Elaphe moellendorffi) and beauty snakes (Elaphe taenuris) that died soon after importation into the USA. Viper heart (VH2) cells inoculated with tissue homogenates showed cytopathic effects consisting of large syncytia formation followed by cell detachment from the monolayer. Tissue culture supernatants failed to hemagglutinate guinea pig and chicken erythrocytes at room temperature. Electron microscopy of purified virions revealed spherical to icosahedral particles measuring 70-85 nm in diameter with a double capsid layer. Preparations of the viral genome contained ten segments of dsRNA when analyzed by polyacrylamide gel electrophoresis. A juvenile black ratsnake (Elaphe obsoleta obsoleta) was experimentally inoculated with the isolate and was found dead 26 days post inoculation. Necropsy revealed diffuse subacute interstitial pneumonia with respiratory epithelial cell hyperplasia and syncytia. Reovirus isolated from this snake was used to inoculate another juvenile black ratsnake which was euthanized 40 days post inoculation. Pneumonia and multifocal subacute proliferative tracheitis were found on necropsy. Reovirus was isolated from the lung of this snake and was demonstrated by transmission electron microscopy. This is the first documentation of a pathogenic reptile reovirus and the first report of experimental transmission of a reovirus in snakes.

Targeted disruption of the Cln3 gene provides a mouse model for Batten disease. The Batten Mouse Model Consortium [corrected].

Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease.

Ultrastructural characterization of the agent of systemic yeast infection of owl monkeys.

Systemic infection by an unclassified yeast-like organism has been encountered sporadically in wild-caught owl monkeys (Aotus sp.) from South America. The infection is presumably acquired in the wild; the incubation period ranges from months to years. The disease is indolent and clinical signs are non-specific. The diagnosis is based on histopathologic observation of yeast-like cells in multiple internal organs. Most cells appear to be phagocytized by macrophages, however, many are apparently free in the extracellular space. Other inflammatory infiltrates, including neutrophils, lymphocytes, plasma cells, and multinucleated giant cells, are conspicuously absent. Cells are thick-walled, globose to oval, range from 5 to 8 micron in diameter, and reproduce by narrow-based budding of single daughter cells. Attempts to cultivate the organism on artificial media have failed. Yeast cell ultrastructure was studied using transmission electron microscopy. The cell wall is multilayered, and the internal structure is markedly heterogenous. In some cells, the cytoplasm is lightly electron-opaque, finely granular and lacks recognizable organelles or nuclei. In others, the cytoplasm is electron dense and contains mitochondria, ribosome-like granules, and a multilobulated nucleus. This organism differs from other recognized pathogenic yeast in its combined light microscopic appearance, organ involvement and host response. Ultrastructurally, it most closely resembles Loboa loboi.

Rate allocation for spotlight SAR phase history data compression.

Complex phase history data in synthetic aperture radar (SAR) systems require extensive processing before useful images can be obtained. In spotlight mode SAR systems, useful images can be obtained by applying aperture weighting and inverse Fourier transform operations to SAR phase history data. In this paper, we are concerned with the compression of the complex phase history data obtained by a spotlight SAR system. We exploit knowledge of the aperture weighting function along with Fourier transform processing to attach a "gain" factor to each complex phase history data sample. This gain factor is then used to efficiently allocate bits to the phase history data during quantization. Performance evaluations are presented for this compression system relative to other existing SAR phase history data compression systems.

c-Met autocrine activation induces development of malignant melanoma and acquisition of the metastatic phenotype.

The molecular and genetic events that contribute to the genesis and progression of cutaneous malignant melanoma, a complex and aggressive disease with a high propensity for metastasis, are poorly understood due in large part to the dearth of relevant experimental animal models. Here we used transgenic mice ectopically expressing hepatocyte growth factor/scatter factor (HGF/SF) to show that the Met signaling pathway is an important in vivo regulator of melanocyte function, whose subversion induces malignant melanoma. Tumorigenesis occurred in stages, beginning with the abnormal accumulation of melanocytes in the epidermis and dermis and culminating in the development of metastatic melanoma. Oncogenesis in this model was driven by creation of HGF/SF-Met autocrine loops through forced expression of the transgenic ligand and apparent selection of melanocytes overexpressing endogenous receptor, rather than paracrine stimulation or mutational activation of c-met. Preference for liver as a metastatic target correlated with high HGF/SF-Met autocrine activity, consistent with the notion that such activity may influence colonization. Although basic fibroblast growth factor and its receptor were both weakly expressed in the majority of melanomas examined, high levels were found only in those rare neoplasms with low or undetectable HGF/SF and Met expression, suggesting that these two tyrosine kinase receptor autocrine loops serve a critical overlapping function in melanocytic tumorigenesis. Our data support a causal role for HGF/SF-Met signaling in the development of melanoma and acquisition of the metastatic phenotype. Moreover, this transgenic mouse should serve as a highly useful model, facilitating our understanding of mechanisms by which human melanoma progresses to malignancy and expediting the development of efficacious therapeutic modalities designed to constrain metastasis.

Chromatographic analysis of photodynamically significant porphyrin dimers and trimers.

Two porphyrin dimers, dihematoporphyrin dimer (DHD) and divinyl dimer (DVD), and two porphyrin trimers, dihematoporphyrin trimer (DHT) and divinyl trimer (DVT), have been analyzed utilizing isocratic ion-pair reversed-phase high-performance liquid chromatography. Results indicate that the vinyl porphyrins can be distinguished by three peaks appearing near 15, 38, and 42 min. The hematoporphyrin complexes are identified by the appearance of a peak located at 35 min. The DVT and DVD complexes present unique chromatographic markers at 28 and 15 min, respectively. Based on the location of these chromatographic markers, it was found that the Photofrin drug must contain the DVD and the DHT complexes, but does not contain the DVT complex. The purity of the DVT complex is compromised by the presence of DHD and DHT impurities.

Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia.

Adenovirus vectors are capable of high efficiency in vivo arterial gene transfer, and are currently in use as therapeutic agents in animal models of vascular disease. However, despite substantial data on the ability of viruses to cause vascular inflammation and proliferation, and the presence in current adenovirus vectors of viral open reading frames that are translated in vivo, no study has examined the effect of adenovirus vectors alone on the arterial phenotype. In a rabbit model of gene transfer into a normal artery, we examined potential vascular cell activation, inflammation, and neointimal proliferation resulting from exposure to replication-defective adenovirus. Exposure of normal arteries to adenovirus vectors resulted in: (a) pronounced infiltration of T cells throughout the artery wall; (b) upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in arterial smooth muscle cells; (c) neointimal hyperplasia. These findings were present both 10 and 30 d after gene transfer, with no evidence of a decline in severity over time. Adenovirus vectors have pleiotropic effects on the arterial wall and cause significant pathology. Interpretation of experimental protocols that use adenovirus vectors to address either biological or therapeutic issues should take these observations into account. These observations should also prompt the design of more inert gene transfer vectors.

Dissociation of synchronization and excitability in furosemide blockade of epileptiform activity.

Furosemide, a chloride cotransport inhibitor, reversibly blocked synchronized burst discharges in hippocampal slices without reducing the pyramidal cell response to single electrical stimuli. Images of the intrinsic optical signal acquired during these slice experiments indicated that furosemide coincidentally blocked changes in extracellular space. In urethane-anesthetized rats, systemically injected furosemide blocked kainic acid-induced electrical discharges recorded from cortex. These results suggest that (i) neuronal synchronization involved in epileptiform activity can be dissociated from synaptic excitability; (ii) nonsynaptic mechanisms, possibly associated with furosemide-sensitive cell volume regulation, may be critical for synchronization of neuronal activity; and (iii) agents that affect extracellular volume may have clinical utility as antiepileptic drugs.

Directional variation of spatial and temporal characteristics of limb movements made by monkeys in a two-dimensional work space.

1. The directional variation of kinematic and electromyographic (EMG) characteristics of two-joint arm movements made to targets in a two-dimensional work space was studied in monkeys trained to make targeted arm movements under different behavioral conditions. 2. In each animal, kinematic measures of movement (movement amplitude, movement time, peak velocity, and trajectory curvature) and endpoint spatial position within the target zone varied as a function of the direction of the target from the starting position. Movements made toward the body into the ipsilateral hemispace generally had the smallest amplitude, lowest peak velocity, and longest movement time. 3. Although the directional variation in peak velocity could partially be accounted for by predicted anisotropies in the inertial load imposed by the arm, deviations from these predictions suggest that movement amplitude is controlled more rigorously by the CNS. Adjustments in movement time may be used to compensate for inertial anisotropies. 4. The spatial characteristics of movements (amplitude, trajectory curvature, or endpoint error) were influenced little by the visibility of the target during movement, the advanced knowledge of target location, or the presence or absence of an external trigger cue. However, temporal characteristics (movement time, peak velocity, and for some animals, reaction time) varied more as sensory cues were changed. 5. The time of initial EMG activity in muscles acting around the shoulder varied systematically as a function of target direction. A cosine model accounted for a large fraction of the variability in initial onset time, as determined in a trial-by-trial analysis. The amplitude of the EMG activity was more narrowly tuned, however. Muscles acting at the elbow showed less activity and more variable directional tuning. 6. We conclude that directional variations in the kinematic characteristics of movement, and thus, the dynamic force requirements of the task, must be taken into consideration as contributors to the apparent directional coding described for neuronal populations in different portions of the CNS.

Isolation and photodynamic effects of hematoporphyrin derivative components: a chromatographic analysis of the starting materials.

Twenty different fractions of hematoporphyrin derivatives (HpD) and eight fractions of an HpD dimer mixture were isolated utilizing isocratic reversed-phase ion-pair high-performance liquid chromatography. These fractions were characterized by UV-visible and fluorescence spectrophotometry. Fluorescence quantum yields and photokill efficiency for each fraction in PTK2 epithelial cells were obtained. Results indicate that some part of the photoactivity exhibited by HpD may be due to impurities present in the HpD starting material, hematoporphyrin-IX dihydrochloride, depending on its source. It was also found that hematoporphyrin D, a commercial acetylated product formed during synthesis of HpD, contained a higher percentage of monomers than would be expected.

Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators.

To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the alpha isoform of mouse PPAR (mPPAR alpha) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR alpha protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional-activation of target genes were not observed. These results clearly demonstrate that mPPAR alpha is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR alpha-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.

bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.

HIV-associated nephropathy is characterized by extensive tubulointerstitial disease with epithelial cell injury, microcystic proliferation, and tubular regeneration with glomerulosclerosis. To explore the role of bFGF as a mediator of HIV-induced interstitial disease, we utilized an HIV transgenic mouse model that manifests clinical and histological features observed in patients. In transgenic mice, simultaneous renal epithelial cell proliferation and injury were detected in vivo. In areas of microcystic proliferation, immunoreactive bFGF colocalized with extracellular matrix. Kidneys from transgenic mice had increased bFGF low affinity binding sites, particularly in the renal interstitium. In vitro, transgenic renal tubular epithelial cells proliferated more rapidly and generated tubular structures spontaneously, in marked contrast to nontransgenic renal cells where these pathologic features could be mimicked by exogenous bFGF. These studies suggest that renal bFGF and its receptors play an important role in the pathogenesis of HIV-associated nephropathy.

Environmental monitoring of bleached kraft pulp mill chlorophenolic compounds in a northern Canadian river system.

The environmental transport of pulp mill effluent compounds and the exposure of two fish species has been monitored by parallel analyses of effluent, water column and suspended sediment samples, and fish bile and muscle. Compounds analyzed included over 20 chlorophenolic compounds and 12 fatty and resin acids. The concentration of chlorophenols varied with seasonal river flows and mill process changes such as the substitution of chlorine dioxide (ClO2) for chlorine gas (Cl2) in the bleach plant. At 100% (ClO2) substitution, the effluent and the water column concentrations of most chlorophenolics approached the analytical detection limits of 0.1-1 parts per billion. Chlorophenolic and fatty/resin acid compounds were detected in the bile of both mountain whitefish (Prosopium williamsoni) and longnose sucker (Catostomus catostomus), but were rarely detected in fillets. Fish bile concentrations were observed in an apparent spatial gradient as far as 230 km downstream of the mill. A depuration experiment with fish held in uncontaminated water for eight days indicated a rapid decrease in chlorophenol levels. These observations corroborate previous investigations that chlorophenolic compounds are rapidly excreted and can be used as sensitive markers for recent exposure to mill effluents.

Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice.

In earlier studies using a streptomycin-treated mouse model of infection caused by enterohemorrhagic Escherichia coli (EHEC), animals fed Shiga-like toxin type II (SLT-II)-producing strains developed acute renal cortical necrosis and died, while mice fed Shiga-like toxin type I (SLT-I)-producing clones did not die (E. A. Wadolkowski, L. M. Sung, J. A. Burris, J. E. Samuel, and A. D. O'Brien, Infect. Immun. 58:3959-3965, 1990). To examine the bases for the differences we noted between the two toxins in the murine infection model, we injected mice with purified toxins and carried out histopathological examinations. Despite the genetic and structural similarities between the two toxins, SLT-II had a 50% lethal dose (LD50) which was approximately 400 times lower than that of SLT-I when injected intravenously or intraperitoneally into mice. Histopathologic examination of toxin-injected mice revealed that detectable damage was limited to renal cortical tubule epithelial cells. Passive administration of anti-SLT-II antibodies protected mice from SLT-II-mediated kidney damage and death. Immunofluorescence staining of normal murine kidney sections incubated with purified SLT-I or SLT-II demonstrated that both toxins bound to cortical tubule and medullary duct epithelial cells. Compared with SLT-I, SLT-II was more heat and pH stable, suggesting that SLT-II is a relatively more stable macromolecule. Although both toxins bound to globotriaosylceramide, SLT-I bound with a higher affinity in a solid-phase binding assay. Differences in enzymatic activity between the two toxins were not detected. These data suggest that structural/functional differences between the two toxins, possibly involving holotoxin stability and/or receptor affinity, may contribute to the differential LD50s in mice.