Failure-to-thrive syndrome associated with tumor formation by Madin-Darby canine kidney cells in newborn nude mice.

Tumors that formed in newborn nude mice that were inoculated with 10(7) Madin-Darby canine kidney (MDCK) cells were associated with a failure-to-thrive (FTT) syndrome consisting of growth retardation, lethargy, weakness, and dehydration. Scoliosis developed in 41% of affected pups. Pups were symptomatic by week 2; severely affected pups became moribund and required euthanasia within 3 to 4 wk. Mice with FTT were classified into categories of mild, moderate, and severe disease by comparing their weight with that of age-matched normal nude mice. The MDCK-induced tumors were adenocarcinomas that invaded adjacent muscle, connective tissue, and bone; 6 of the 26 pups examined had lung metastases. The induction of FTT did not correlate with cell-line aggressiveness as estimated by histopathology or the efficiency of tumor formation (tumor-forming dose 50% endpoint range = 10(2.8) to 10(7.5)); however, tumor invasion of the paravertebral muscles likely contributed to the scoliosis noted. In contrast to the effect of MDCK cells, tumor formation observed in newborn mice inoculated with highly tumorigenic, human-tumor-derived cell lines was not associated with FTT development. We suggest that tumor formation and FTT are characteristics of these MDCK cell inocula and that FTT represents a new syndrome that may be similar to the cachexia that develops in humans with cancer or other diseases.

Anthrax lethal toxin disrupts intestinal barrier function and causes systemic infections with enteric bacteria.

A variety of intestinal pathogens have virulence factors that target mitogen activated protein kinase (MAPK) signaling pathways, including Bacillus anthracis. Anthrax lethal toxin (LT) has specific proteolytic activity against the upstream regulators of MAPKs, the MAPK kinases (MKKs). Using a murine model of intoxication, we show that LT causes the dose-dependent disruption of intestinal epithelial integrity, characterized by mucosal erosion, ulceration, and bleeding. This pathology correlates with an LT-dependent blockade of intestinal crypt cell proliferation, accompanied by marked apoptosis in the villus tips. C57BL/6J mice treated with intravenous LT nearly uniformly develop systemic infections with commensal enteric organisms within 72 hours of administration. LT-dependent intestinal pathology depends upon its proteolytic activity and is partially attenuated by co-administration of broad spectrum antibiotics, indicating that it is both a cause and an effect of infection. These findings indicate that targeting of MAPK signaling pathways by anthrax LT compromises the structural integrity of the mucosal layer, serving to undermine the effectiveness of the intestinal barrier. Combined with the well-described immunosuppressive effects of LT, this disruption of the intestinal barrier provides a potential mechanism for host invasion via the enteric route, a common portal of entry during the natural infection cycle of Bacillus anthracis.

Heterogeneity of the tumorigenic phenotype expressed by Madin-Darby canine kidney cells.

The mechanisms by which cells spontaneously immortalized in tissue culture develop the capacity to form tumors in vivo likely embody fundamental processes in neoplastic development. The evolution of Madin-Darby canine kidney (MDCK) cells from presumptively normal kidney cells to immortalized cells that become tumorigenic represents an example of neoplastic development in vitro. Studies of the mechanisms by which spontaneously immortalized cells develop the capacity to form tumors would benefit from quantitative in vivo assays. Most mechanistic correlations are evaluated by using single-dose tumor-induction experiments, which indicate only whether cells are or are not tumorigenic. Here we used quantitative tumorigenicity assays to measure dose-and time-dependent tumor development in nude mice of 3 lots of unmodified MDCK cells. The results revealed lot-to-lot variations in the tumorigenicity of MDCK cells, which were reflected by their tumor-inducing efficiency (threshold cell dose represented by mean tumor-producing dose; log(10) 50% endpoints of 5.2 for vial 1 and 4.4 for vial 2, and a tumor-producing dose of 5.8 for vial 3) and mean tumor latency (vial 1,6.6 wk; vial 2,2.9 wk; and vial 3,3.8 wk). These studies provide a reference for further characterization of the MDCK cell neoplastic phenotype and may be useful in delineating aspects of neoplastic development in vitro that determine tumor-forming capacity. Such data also are useful when considering MDCK cells as a reagent for vaccine manufacture.

Neutrophil elastase mediates pathogenic effects of anthrax lethal toxin in the murine intestinal tract.

Neutrophils isolated from BALB/c or C57BL/6 mice and treated in vitro with anthrax lethal toxin release bioactive neutrophil elastase, a proinflammatory mediator of tissue destruction. Similarly, neutrophils isolated from mice treated with anthrax lethal toxin in vivo and cultured ex vivo release greater amounts of elastase than neutrophils from vehicle-treated controls. Direct measurements from murine intestinal tissue samples demonstrate an anthrax lethal toxin-dependent increase in neutrophil elastase activity in vivo as well. These findings correlate with marked lethal toxin-induced intestinal ulceration and bleeding in neutrophil elastase(+/+) animals, but not in neutrophil elastase(-/-) animals. Moreover, neutrophil elastase(-/-) mice have a significant survival advantage over neutrophil elastase(+/+) animals following exposure to anthrax lethal toxin, thereby establishing a key role for neutrophil elastase in mediating the deleterious effects of anthrax lethal toxin.

Serotonergic influences on life-history outcomes in free-ranging male rhesus macaques.

Several studies have demonstrated that nonhuman primate males with low cerebrospinal fluid (CSF) levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) exhibit antisocial behavior patterns. Included in these deleterious patterns are impulse control deficits associated with violence and premature death. No studies to date have longitudinally studied the long-term outcome of young subjects with low CSF 5-HIAA concentrations as they mature into adults. In this study we examined longitudinal relations among serotonergic and dopaminergic functioning, as reflected in CSF metabolite concentrations, aggression, age at emigration, dominance rank, and mortality in free-ranging rhesus macaque (Macaca mulatta) males. Our results indicate long-term consistency of individual differences in levels of 5-HIAA in CSF in the subject population from the juvenile period of development through adulthood. We found a significant negative correlation between 5-HIAA concentrations measured in juveniles and rates of high-intensity aggression in the same animals as adults. Further, CSF 5-HIAA concentrations were lower in juveniles that died than in animals that survived. For the young animals that migrated there was a positive correlation between CSF 5-HIAA concentration and age at emigration, whereas for the animals that remained in their troop until later in sexual maturity there was a negative correlation between CSF 5-HIAA concentration and age of emigration. After animals emigrated to a new troop, social dominance rank in the new troop was positively correlated with early family social dominance rank, but inversely correlated with juvenile CSF 5-HIAA concentrations. Taken together, our findings suggest that males with low central serotonin levels early in life delay migration and show high levels of violence and premature death, but the males that survive achieve high rank. These findings indicate that longitudinal measures of serotonergic and dopaminergic functioning are predictive of major life-history outcomes in nonhuman primate males. Low concentrations of CSF 5-HIAA are associated with negative life-history patterns characterized by social instability and excessive aggression, and positive life-history patterns characterized by higher dominance rank.

Left versus right nipple preference in free-ranging infant rhesus macaques (Macaca mulatta).

The examination of nonhuman primate (NHP) lateralized behaviors may provide insight into the evolution of hemispheric specialization. This study examined nipple preference in 64 infant macaques in order to consider the ontogeny of lateralized behavior. We used a focal animal sampling method to record nipple contact during 15, 30-min observation sessions collected across each infant's first year of life. Using a lateralized behavior index (LBI) we calculated individual and population preferences (LBI=(R-L)/(R+L); "R"=mean right nipple contact, "L"=mean left nipple contact). Strength of preference was calculated as the absolute value of this score. Infants exhibited no population preference for a particular nipple, but showed a significant strength of preference that developed after 48 hr. Interestingly, successive siblings preferred the nipple not used by the previous infant. These findings suggest that nipple preference is guided by external stimuli, and that nipple preference during infancy may not be a behavioral representation of hemispheric specialization.

Physiological correlates of aggression and impulsivity in free-ranging female primates.

We examined the relations among cerebrospinal fluid (CSF) monoamine metabolite concentrations, plasma hormone concentrations, aggression, and impulsive risk-taking behavior in a free-ranging population of female rhesus macaques. We selected 44 juvenile female rhesus macaques as subjects from a population of approximately 3000 macaques that inhabit a 475-acre Sea Island. We obtained CSF and blood samples, and recorded behavioral observations over a subsequent 18-month period. Our results indicate an inverse correlation between CSF concentrations of the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), and the frequency of low-intensity restrained aggression typically associated with matrilineal defense of social status. In contrast, previous research with males has shown an inverse correlation between CSF 5-HIAA concentrations and levels of violent unstrained aggression typically associated with traumatic injury and death. We also noted a negative correlation between plasma concentrations of the stress hormone cortisol and the frequency of low-intensity aggressive acts, a finding not reported in our previous studies with males. Further examination revealed a negative correlation between CSF 5-HIAA concentrations and the rate of long dangerous leaps through the forest canopy, suggesting that the relation between low serotonergic functioning and impulsivity may generalize to both female and male primates. These results indicate that females with low CSF 5-HIAA concentrations, like their male counterparts, are at increased risk for impulsive temperament, but that unlike males, females may be buffered from this risk through intersexual differences in life history patterns and social affiliation.