Glucocorticoid resistance in T-lineage acute lymphoblastic leukaemia is associated with a proliferative metabolism.

Glucocorticoids (GCs) are among the most important drugs for acute lymphoblastic leukaemia (ALL), yet despite their clinical importance, the exact mechanisms involved in GC cytotoxicity and the development of resistance remain uncertain. We examined the baseline profile of a panel of T-ALL cell lines to determine factors that contribute to GC resistance without prior drug selection. Transcriptional profiling indicated GC resistance in T-ALL is associated with a proliferative phenotype involving upregulation of glycolysis, oxidative phosphorylation, cholesterol biosynthesis and glutamate metabolism, increased growth rates and activation of PI3K/AKT/mTOR and MYC signalling pathways. Importantly, the presence of these transcriptional signatures in primary ALL specimens significantly predicted patient outcome. We conclude that in lymphocytes the activation of bioenergetic pathways required for proliferation may suppress the apoptotic potential and offset the metabolic crisis initiated by GC signalling. It is likely that the link between GC resistance and proliferation in T-ALL has not been fully appreciated to date because such effects would be masked in the context of current multiagent therapies. The data also provide the first evidence that altered expression of wild-type MLL may contribute to GC-resistant phenotypes. Our findings warrant the continued development of selective metabolic inhibitors for the treatment of ALL.

Authenticity and drug resistance in a panel of acute lymphoblastic leukaemia cell lines.

Cell lines are important models for drug resistance in acute lymphoblastic leukaemia (ALL), but are often criticised as being unrepresentative of primary disease. There are also doubts regarding the authenticity of many lines. We have characterised a panel of ALL cell lines for growth and drug resistance and compared data with that published for primary patient specimens. In contrast to the convention that cell lines are highly proliferative, those established in our laboratory grow at rates similar to estimates of leukaemic cells in vivo (doubling time 53-442 h). Authenticity was confirmed by genetic fingerprinting, which also demonstrated the potential stability of long-term cultures. In vitro glucocorticoid resistance correlated well with that measured ex vivo, but all lines were significantly more sensitive to vincristine than primary specimens. Sensitivity to methotrexate was inversely correlated to that of glucocorticoids and L-asparaginase, indicating possible reciprocity in resistance mechanisms. A cell line identified as highly methotrexate resistant (IC50 > 8000-fold higher than other lines) was derived from a patient receiving escalating doses of the drug, indicating in vivo selection of resistance as a cause of relapse. Many of these lines are suitable as models to study naturally occurring resistance phenotypes in paediatric ALL.

Risk of disease spread through bioterrorism.

Bioterrorism is seen as a clear and present danger, although historically, acts of bioterrorism have been relatively unpredictable, rare and, thus far, small-scale events. The risk of an event is elevated by increasing contact among species and a global connectivity that provides rapid dissemination of infectious diseases regardless of origin. Virtually any pathogenic microbe could be used by bioterrorists. An attack may be difficult to distinguish from a naturally occurring infectious disease outbreak; however, consequences are likely to be similar. The agricultural sector is extremely vulnerable to bioterrorist attacks because animals and plants have little or no innate resistance to foreign pathogens and are not vaccinated or otherwise protected against these diseases. It is also important to note that weapons or delivery systems are not an issue because the animals and plants themselves are the primary vector for transferring agents. Many bioterrorism agents are zoonotic in origin, thus an attack on animal populations could pose a health risk to humans. Additionally, disease outbreaks resulting from bioterrorism could jump to wildlife species, persist in the environment, replace locally adapted enzootic strains, expand their range, or emerge as a new zoonotic disease in naïve human and animal populations.

Spontaneous terminal ileitis resembling Crohn disease in captive tamarins.

Five tamarins (four Saguinus mystax and one S. labiatus) died with wasting syndrome characterized by chronic diarrhea at the Center for Reproduction and Conservation of Non-Human Primates in Iquitos, Peru. At necropsy, the terminal ileum of all affected tamarins was found to be markedly thickened. Histologically, the terminal ileal mucosa was completely ulcerated, and effaced by debris and mononuclear inflammatory cells. The submucosa and serosa were thickened by fibroplasia, mononuclear cell infiltrates and variable edema. No infectious agent was observed. The lesions were similar to those described previously for Crohn disease. This is to our knowledge the first report of terminal ileitis resembling Crohn disease in non-human primates.

Intrinsic connections in the caudal subdivision of the dorsolateral visual area (DL(C)) in squirrel monkeys.

Patterns of intrinsic connections and features of individual intrinsic axons in the caudal subdivision of the dorsolateral visual area (DL(C)) were investigated in four squirrel monkeys (Saimiri) following extracellular injections of the tracers biocytin, biotinylated dextran amine, and wheat germ agglutinin conjugated to horseradish peroxidase. Injections were defined in DL(C) by reference to architectonic borders and patterns of connections with other cortical areas. Intrinsic connections extended up to 6 mm from an injection and were usually anisotropic, extending farther dorsoventrally than anteroposteriorly. Injections that involved the supragranular layers produced up to 20 mainly supragranular patches of projections that had a width of 285 +/- 8 microm (mean +/- standard error) and area of 0.125 +/- 0.016 mm(2). Seventy-four intrinsic axon segments with a total of 3,290 boutons were drawn and their bouton spacing measured. The sample included axons in layers 1, 2-3, 5, and multiple (>2) layers; horizontally and vertically oriented axons; and axons in an injection halo, patch, or nonpatch region of projections. There were no differences in bouton spacing for axons in halo, patch, or nonpatch regions. Layer 1 axons (n = 7) had a significantly sparser distribution of boutons (median interbouton interval of 45.2 +/- 17.8 microm) than the layers 2-3 (n = 35) and layer 5 axons (n = 26), which did not differ in bouton spacing (median interbouton intervals of 8.1 +/- 0.4 microm and 8.4 +/- 0.8 microm, respectively). Patterns of intrinsic connections in DL(C) are related to neural organization and properties reported for DL or visual area V4, and are compared to intrinsic connections of other areas.

K-ras mutations in 239PuO2 canine lung neoplasms.

Single-strand conformational polymorphism (SSCP) analysis and direct sequencing methods were used to examine lung tumors derived from a cohort of beagle dogs with inhalational exposures to 239PuO2. These exposures were done at Pacific Northwest Laboratories where 18-month-old beagle dogs were given 239PuO2 by single-dose inhalation and allowed to live out their life-spans. Formalin-fixed paraffin-embedded blocks of tissues from 25 dogs exposed to 239PuO2 by aerosol inhalation which later developed lung tumors were available for this study. Two of 25 tumors had mutations within exon 1 of K-ras detected by SSCP analysis. Both mutations were GGT to GAT transitions at codon 12 confirmed by direct sequencing experiments. One was an adenocarcinoma from the medium-high exposure group and the other was a broncheolo-alveolar carcinoma from the medium-low exposure group. The rate of K-ras mutations in plutonium-induced lung tumors described herein (8%) was greater than previously described in canine plutonium-induced lung tumors (0%), but was less than that which we have described in spontaneous canine lung cancer (16%), less than that reported for human spontaneous non-small cell lung cancer (13-36%) and less than that described in rats with spontaneous lung cancer (40%) or lung tumors following 239Pu inhalation exposure (46%).

Biological effects of inhaled 238PuO2 in beagles.

Beagle dogs exposed to 238PuO2 aerosols (136 dogs, 13-22 per group, mean initial lung depositions of 0.0, 0.13, 0.68, 3.1, 13, 52 and 210 kBq) were observed throughout life to determine tissues at risk and dose-effect relationships. The pulmonary retention of 238Pu was represented by the sum of two exponentially decreasing components of the initial lung deposition; about 84% cleared with a 174-day half-time; the half-time of the remainder was 908 days. The average percentages of final body burden found in lung, skeleton, liver and thoracic lymph nodes in the 30 longest-surviving dogs (mean survival 14 years) were 1, 46, 42 and 6%, respectively. Of 116 beagles exposed to plutonium, 34 (29%) developed bone tumors, 31 (27%) developed lung tumors, and 8 (7%) developed liver tumors. Although lungs accumulated a higher average radiation dose than skeleton, more deaths were due to bone tumors than to lung tumors. Deterministic effects included radiation pneumonitis, osteodystrophy, hepatic nodular hyperplasia, lymphopenia, neutropenia and sclerosing tracheobronchial lymphadenitis. Hypoadrenocorticism was also observed in a few dogs. Increased serum alanine aminotransferase, indicative of liver damage, was observed in groups with > or =3.1 kBq initial lung deposition. Estimates of cumulative tissue dose in a human exposed to airborne 238PuO2 for 50 years at a rate of one annual limit on intake each year were derived based on a comparison of the data on metabolism for humans and beagles. The 50-year dose estimates for humans are an order of magnitude lower than doses at which increased incidence of neoplasia was observed in these dogs, whereas the projected doses to humans from 50-year exposure at the annual limit of intake are of similar magnitude to those at which deterministic effects were seen in the beagles.

Reproduction of the owl monkey (Aotus spp.) in captivity.

The reproduction performance of captive owl monkeys, a breed used extensively in biomedical research, was observed at the Battelle Primate Facility (BPF). The colony grew through captive breeding, imports from the Peruvian Primatological Project, and others to a peak size of 730. It included seven karyotypes of Aotus sp. Results showed that owl monkeys can breed successfully in a laboratory in numbers sufficient to sustain modest research programs. Reproductive success increases when pairs are compatible, of the same karyotype, and stabilized; however, mated pairs of different karyotype are also productive. Under conditions of controlled lighting and heating, owl monkeys at BPF showed no birth peak nor birth season.

Hypoadrenocorticism in beagles exposed to aerosols of plutonium-238 dioxide by inhalation.

Hypoadrenocorticism, known as Addison's disease in humans, was diagnosed in six beagles after inhalation of at least 1.7 kBq/g lung of 238PuO2. Histological examination of adrenal gland specimens obtained at necropsy revealed marked adrenal cortical atrophy in all cases. Autoradiographs showed only slight alpha-particle activity. Although the pathogenesis of adrenal cortical atrophy in these dogs is unclear, there is evidence to suggest an autoimmune disorder linked to damage resulting from alpha-particle irradiation to the lymphatic system.

The distribution and effects of inhaled 239Pu(NO3)4 deposited in the liver of dogs.

The distribution and effects of inhaled 239Pu(NO3)4 deposited in the liver of dogs were studied in five groups of 20 beagles exposed to initial lung depositions ranging from 1.0 to 520 Bq g(-1) lung. Following life-span observations, the liver contained 40 +/- 1% of the final body deposition of plutonium, second only to the skeleton. The liver-to-skeleton ratio of deposited plutonium for total organ was 0.8, or 3.5 when expressed on a per-gram basis. There was no effect of exposure level on liver-to-skeleton ratios. Autoradiographs showed that the dose rate delivered to parenchymal cells was higher than evident from radiochemical analysis of the whole organ. Elevated levels of serum liver enzymes were observed in groups with mean liver concentrations of 1.3 Bq g(-1) and liver doses of 3 Gy or higher. Nodular hyperplasia of liver and bile-duct hyperplasia were observed. Liver tumors, principally of bile-duct epithelium, were late-occurring and were observed at lower exposure levels at which life span was not shortened by lung or bone tumors.

Serum and urine biochemical diversity among adult wild-caught Aotus nancymae and Saimiri peruviensis.

Serum and urine analytes were compared between adult wild-caught owl monkeys (Aotus nancymae) and adult wild-caught squirrel monkeys (Saimiri peruviensis) to determine if normative clinical pathology data were similar. An objective of the study was to confirm that species of neotropical primates are distinct with regard to physiologic parameters, and should not be considered interchangeable in biomedical research. Significant differences (P < 0.05) were noted in many serum and urine analytes between the two groups. The results suggest that reference data for wild-caught owl monkeys are not applicable to squirrel monkeys, and the differences are sufficiently large to be of clinical significance. These findings illuminate the diversity among species of neotropical primates.

Dietary induction of cholesterol gallstones in the owl monkey: preliminary findings in a new animal model.

The owl monkey (Aotus nancymae) is a primate with a bile acid and biliary lipid profile resembling that of humans. Aotus spp. are among the rare species, including humans, that spontaneously develop cholesterol gallstones. With dietary induction the owl monkey proved a rapid, reliable model of cholesterol cholelithiasis. Six owl monkeys, three of each sex, were fed a diet supplemented with 1.5% cholesterol for 5 weeks. Each week blood samples were drawn for cholesterol determination, and bile samples were obtained by ultrasound-guided percutaneous aspiration of the gallbladder. Weekly ultrasound imaging documented development of gallbladder sludge in all animals, with eventual stone formation in five of six. At necropsy after 5 weeks consuming the diet, all animals had distinct sludging and/or small stones in the gallbladder, correlating with the ultrasound findings. Plasma cholesterol values remained lower in females but increased markedly in some males to > 1,400 mg/dl. Histologic examination revealed mild, diffuse hepatocellular lipidosis and degeneration in four of six animals. Detailed examination of the gallbladder indicated that transhepatic needle punctures induced minimal focal abnormalities, judged inconsequential. In contrast to rodent models commonly in use, owl monkeys have liver and digestive tract anatomy and bile physiology that is similar to that in humans. These similarities give this model the potential to substantively improve understanding of the pathogenesis, treatment, and prevention of human cholesterol gallstones. This model can provide sequential, simultaneous correlation of plasma and biliary lipids, imaging of gallbladder contents, and physiologic processes.

Hepatic effects of inhaled plutonium dioxide in beagles.

The effects of inhaled 238PuO2 deposited in the liver of dogs were studied in beagles exposed to initial lung depositions ranging from 5.7 to 2979.7 Bq/g lung. Approximately 20% of the initial lung deposition was translocated to the liver by 1500 days after exposure. Life-span observations revealed that the liver contained 40% of the final body burden of plutonium, second only to the skeleton. Elevated serum liver enzyme activities were observed in dogs with final liver depositions of > or = 0.4 Bq/g, cumulative dose to the liver of > or = 0.18 Gy and annual dose rate > or = 0.02 Gy/year. Enzyme elevations were seen at one dose level lower than that in which bone or lung tumors were observed. Linear regression analysis revealed strong to moderate correlation between cumulative dose and dose rate and time to observed increases in liver enzyme activities. Liver tumors were late-occurring neoplasms observed at lower exposure levels where life span was not shortened by lung and bone tumors.

Hematologic values of the wild-caught karyotype V owl monkey (Aotus vociferans).

Blood samples from 35 wild-caught owl monkeys (Aotus vociferans, karyotype V) were analyzed to estimate reference intervals of hematologic variables for the species. Findings indicated that distributions were abnormal for leukocytes, neutrophils, eosinophils, basophils, and monocytes. Statistically significant sex differences, too small to be of clinical importance, were found in values for mean cell hemoglobin concentration, leukocytes, and basophils. Reference intervals for the hematologic variables were calculated by the nonparametric percentile estimation method. Comparison of hematologic reference values for wild-caught A. vociferans with those of A. nancymae revealed statistically significant differences for packed cell volume, mean cell volume, leukocytes, eosinophils, basophils, and platelets. These differences also appeared too small to be of clinical significance.

Qualitative and quantitative features of axons projecting from caudal to rostral inferior temporal cortex of squirrel monkeys.

On the basis of cortical and subcortical connections and architectonics, inferior temporal (IT) cortex of squirrel monkeys consists of a caudal region, ITC, with dorsal (ITCd) and ventral (ITCv) subdivisions; a rostral region, ITR; and possibly a third region intermediate to ITC and ITR, ITI (Weller & Steele, 1992; Steele & Weller, 1993). The present study qualitatively and quantitatively examined the terminal arborizations of 26 axons in ITR and ITI labeled by injections of biocytin or, in one case, horseradish peroxidase, in ITCv. The majority of axons gave rise to a single terminal arbor, with a small number branching into two overlapping or nearby arbors. Presumptive terminal specializations consisted of rounded, bead-like swellings, most often located en passant. All axons terminated in layer 4 of cortex, and most had additional terminations in layers 3 and 5. The total extent of each axon's terminal arbor was 125-750 microns dorsoventrally (mean = 360.6 microns) and 150-725 microns anteroposteriorly (mean = 328.1 microns; all values uncorrected for shrinkage). In most axons, especially those with larger terminal fields, boutons were not uniformly distributed, but formed 2-4 clumps (mean = 2.2), with a mean width of 149 microns, separated by narrower regions of fewer boutons. Based on a cluster analysis of characteristics of the 26 axons, axons projecting from caudal (ITCv) to rostral (ITR or ITI) IT cortex of squirrel monkeys comprised three groups that we called Type I, Type II, and Type III. Type I axons, the smallest in area extent of terminal arbor, terminated predominantly in dorsal ITR. Type III axons, largest in areal extent, and Type II axons, intermediate in areal extent, terminated in ventral ITR and throughout ITI. The three classes of axons may correspond to different types of visual information entering rostral IT cortex. The clumping of boutons suggests that individual axons terminate in limited patches within their terminal fields.

Examination of testicular tumours in the beagle dog exposed to inhaled plutonium.

Gross and light microscopic features of testicular neoplasms were examined in the male beagle dog used in three studies to examine the life-span effects of inhaled plutonium (Pu). One hundred and sixty-six cases of testicular neoplasia (TN) occurred among 105 dogs that ranged in age from 7.5 to 17.7 years at the time of diagnosis. The 166 cases of TN comprised 113 interstitial cell tumours, 27 seminomas in situ, 19 seminomas, and seven Sertoli cell tumours. Serum testosterone and estradiol 17-beta concentrations, and the serum testosterone-to-oestradiol ratio were determined in 39 dogs with TN and in five clinically normal, sexually intact, age-matched cohorts. Serum hormone concentrations did not differ significantly among tumour types or between dogs with neoplasms and age-matched cohorts. There was a significant relationship between initial lung deposition (ILD) of Pu and activity in the testis (Bq/g testis). The slope of the relationship was 0.35, 0.89 and 0.91 for 239PuO2, 238PuO2 and 239Pu(NO3)4 respectively. Pu in the testis at long times (> 5 years) after inhalation was between 0.0001 and 0.03% ILD, depending on the physicochemical form of Pu. Although the mean activity of Pu in the testis of dogs was higher in those life-span studies employing 238PuO2 and 239Pu(NO3)4, the cumulative proportion of dogs with tumours, the distribution of tumour types, and mean time to first tumour was not significantly different among the three studies or dose groups, including controls, within a study.

Hematological effects of inhaled plutonium dioxide in beagles.

A life-span study indicated that plutonium activity in the thoracic lymph nodes is a contributor to development of lymphopenia in beagles exposed to 239PuO2. Significant lymphopenia was found in 67 (58%) beagles given a single nose-only exposure to 239PuO2 to result in mean initial lung depositions ranging from 0.69 to 213.3 kBq. Lymphoid atrophy and sclerosis of the thoracic lymph nodes and lymphopenia were observed in exposure-level groups with initial lung depositions > or = 2.5 kBq. Those dogs with final plutonium concentrations in the thoracic lymph nodes > or = 0.4 kBq/g and dose rates > or = 0.01 Gy/day developed lymphopenia. Marked differences existed between chronically lymphopenic dogs and intermittently lymphopenic dogs with regard to initial lung deposition, time to lymphopenic events and absolute lymphocyte concentrations. Linear regression analysis revealed moderate correlation between reduction in lymphocyte values and initial lung deposition, in both magnitude and time of appearance after exposure. Cumulative dose and dose rate appeared to act together to produce initial effects on lymphocyte populations, while dose rate alone appeared to be responsible for the maintenance and subsequent cycles of lymphopenia seen over the life span. No primary tumors were associated with the thoracic lymph nodes in this study, although 70% of the lymphopenic dogs developed lung tumors.

Ultrasound-guided cholecystocentesis in the owl monkey.

Bile samples were obtained from adult owl monkeys by ultrasound-guided percutaneous cholecystocentesis, using a transhepatic approach. Sampling frequency was once weekly over a 5-week period. Clinical recovery from each procedure was rapid. Animal body weights fluctuated within 22% of baseline over the study period, but maximal weight loss in any animal was less than 3% at the study's end. At necropsy, gross lesions in the liver and gallbladder were minimal. Histologic examination revealed mild focal cholecystitis in the gallbladder of four of six animals, focal pericholecystitis in three of six animals, and foci of gallbladder fibrosis in two animals. Changes were consistent with repeated trauma from the centesis procedure. Gallbladder mucosa was judged normal in all animals. Body weight fluctuations were attributed to a change in diet, part of a concurrent study, rather than to the centesis procedure. Ultrasound-guided cholecystocentesis provides a rapid, minimally traumatic, and safe method for repeated bile sampling in a small nonhuman primate.

Spontaneous colitis cystica profunda in captive tamarins.

Of the 232 tamarins (133 Saguinus mystax and 99 Saguinus labiatus) that died at the Center for Reproduction and Conservation of Nonhuman Primates in Iquitos, Peru from January 1987 to December 1990, 23 monkeys (9.9%) were diagnosed as having chronic colitis. Typically, the cecal and colonic mucosa was greyish and small yellowish cysts, measuring 1-4 mm, were found randomly distributed bulging the mucosa. Microscopically, colitis cystica profunda was diagnosed additionally in six more animals, giving a total of 29 cases (12.5%). This is the first report to our knowledge that describes colitis cystica profunda in a nonhuman primate.