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Immune response to fleas in a wild desert rodent: effect of parasite species, parasite burden, sex of host and host parasitological experience.

We studied immune responses of the jird Meriones crassus to different flea species belonging to the same family. We used jirds maintained in an outdoor enclosure (enclosure; N=18) and parasitized by fleas Xenopsylla conformis mycerini and Xenopsylla ramesis, and also jirds born in the laboratory to previously parasitized mothers (laboratory animals; N=23). We asked (i) whether cross-immunity to different fleas occurs, (ii) whether there is a sex difference in immune responses to flea parasitism and (iii) whether the severity of the immune responses depends on parasite load. In the enclosure animals, immune response to antigen from the unfamiliar flea Synosternus cleopatrae pyramidis did not differ from those to antigens from the familiar fleas. In contrast, laboratory rodents demonstrated no difference in the immune response between S. c. pyramidis antigen and either the phytohemagglutinin treatment or controls, although their responses to antigens of fleas familiar to their mothers (X. c. mycerini and X. ramesis) were significantly higher than those to antigen of S. c. pyramidis and phytohemagglutinin. The results clearly demonstrated that (i) cross-reactivity in rodent responses to different flea species occurred for enclosure but not for laboratory jirds and (ii) immune-naïve animals whose mothers were parasitized by fleas had some degree of immunity against fleas. The only sex difference in immunological parameters was the higher level of circulating immune complexes in females than in males. Only phagocytic activity was affected by flea burden, decreasing with an increase in flea numbers.

Analysis of Variance↗

Histo-anatomy of the proximal femur in rats: impact of ovariectomy on bone mass, structure, and stiffness.

BACKGROUND: Hip fractures are the most devastating consequence of osteoporosis in humans. Since the ovariectomized (ovx) rat is a useful model of estrogen-deficient osteoporosis, the purposes of this study were to describe the histo-anatomical features of the rat hip and to determine changes in the proximal femur induced by ovariectomy to evaluate the use of this skeletal site for future bone studies. METHODS: Changes in body mass and composition and in bone mineral content and density were determined by DEXA at 12 weeks after ovariectomy. Gross and histo-anatomy of the rat hip was studied by light microscopy and histomorphometry. Cancellous and cortical bone changes induced by ovx at the femoral midneck were determine using dynamic, static, and structural histomorphometric techniques. The stiffness of the femoral neck was determined by biomechanical testing, and the results were correlated with histological observations and the histomorphometric data. RESULTS: The bony structures of the rat hip, articular cartilage, and muscular and capsular attachments are very similar to the human. Rats, however, have an active growth plate and a well-vascularized periosteum covering the intracapsular portion of the femoral neck, which is different from the adult humans. Rats in the sham and ovx groups exhibited similar biological variations in the thickness of the femoral neck and epiphyseal bone and cartilaginous composition. Ovariectomy promoted periosteal modeling and induced endocortical and cancellous bone remodeling, with a net loss of bone mass due to excess bone resorption. The ovx-induced increase in resorption resulted in reduced trabecular number, thickness, and endocortico-trabecular connectivity, which likely contributed to less bone stiffness in ovx rats relative to the sham controls. CONCLUSIONS: There are numerous similarities in the structure and histology of the rodent and human hip. Skeletal changes induced by ovariectomy in rats, particularly those at the endocortical surface and in the cancellous bone, are very similar to changes observed in the proximal femur in osteoporotic women. In addition, ovx in the rat had compromised the biomechanical properties at the femoral neck, similar to what occurs in the postmenopausal women. Data presented here confirmed responsiveness of the proximal femur in rat to ovarian hormone deficiency, which appears to be a useful and relevant site to investigate mechanisms and interventions relative to human disease.

Absorptiometry, Photon↗

Modulation of cyclin dependent kinase inhibitor proteins and ERK1/2 activity in allylamine-injured vascular smooth muscle cells.

Chronic oxidative injury by allylamine (AAM) induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. The proliferative advantage of AAM vSMC compared to control cells is maintained with serial passage of the cells and the advantage is nullified when AAM cells are seeded on a collagen substrate. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p27 and p21, and mitogen activated protein (MAP) kinases, ERK1/2, in mediating the proliferative advantage of AAM stressed vSMC over control cells on plastic or collagen substrates. p27 levels in randomly cycling cells were comparable in both cell types irrespective of the substrate. In contrast, basal levels of p21 were 1.9 +/- 0.3 (P < 0.05)-fold higher in randomly cycling AAM cells seeded on plastic compared to controls, a difference that was lost on a collagen substrate. Following G0 synchronization, basal levels of both p27 and p21 were higher in AAM cells seeded on plastic compared to controls (1.7 +/- 0.2 and 2.0 +/- 0.3-fold, respectively, P < 0.05), but these differences were lost upon mitogenic stimulation. Pyrrolidine dithiocarbamate (PDTC) decreased p27 and p21 levels in cycling AAM cells relative to controls in a substrate-dependent manner. AAM cells seeded on plastic exhibited enhanced ERK1/2 activation upon mitogenic stimulation; seeding on collagen nullified this advantage. The duration of ERK1/2 activation was prolonged in AAM cells independently of the seeding substrate. We conclude that substrate-dependent acquisition of proliferative phenotypes following repeated cycles of AAM injury correlates with modulation of the cyclin dependent kinase inhibitors, p27 and p21.

Allylamine↗

Effects of endogenous dopamine on measures of [18F]N-methylspiroperidol binding in the basal ganglia: comparison of simulations and experimental results from PET studies in baboons.

The effect of endogenous dopamine on PET measures of radioligand binding is important to the measurement of receptor density (or availability) and neurotransmitter interactions in vivo. We recently reported that pretreatment with amphetamine, a drug which stimulates dopamine release, significantly reduced NMS binding in the baboon brain as determined by the product lambda k3 derived from the graphical analysis method for irreversible systems (lambda is the ratio of the forward to reverse plasma to tissue transport constants and k3 is proportional to receptor density) (Dewey et al.: Synapse 7:324-327, 1991). The purpose of this work is twofold: to evaluate the sensitivity and stability of the analysis method used for the NMS data and from simulation studies which include the competitive effects of dopamine on NMS binding to predict the effect of dopamine on the in vivo PET experiment. Using a measured plasma [18F]-NMS input function from a control study in a baboon, simulation data was numerically generated explicitly allowing competition between NMS and dopamine in the calculation. This data was analyzed using the same techniques as used for the experimental data and the results were compared to in vitro calculations. The following conclusions were reached: 1) The effect of dopamine on specific binding was found to be greater in vivo than in vitro because the in vitro equilibrium experiment is controlled only by the relative Kd's of tracer and dopamine while the in vivo experiment also depends upon the halftime of tracer in tissue which is controlled by the tissue-to-plasma transport constant; 2) Experimental evidence from rodent studies (Seeman et al.: Synapse 3:96-97, 1989) and the agreement between PET studies (Wong et al.: Science 234:1558-1563, 1986a) and postmortem human studies (Seeman et al.: Science 225:728-731, 1984) in schizophrenics suggest that NMS is not likely to be affected by normal levels of endogenous dopamine. From the calculations reported here the effective in vivo Kd of dopamine for the NMS binding site would have to be on the order of or greater than 100 nM, assuming a synaptic dopamine concentration of 20 nM, in order that this concentration of dopamine have little effect on NMS binding.

Amphetamine↗

Effect of maternal under-nutrition on pup body weight and hypothalamic endocannabinoid levels.

Dietary long-chain polyunsaturated fatty acids are known to influence brain levels of the endocannabinoid anandamide in newborn pigs and mice. Furthermore, endocannabinoids were shown to control pup suckling and body weight in mice, and food intake in adult rodents. Here we determined the effect of maternal under-nutrition during gestation, lactation, or both, on body weight, and on the levels of endocannabinoids and expression of cannabinoid CB1 receptors and fatty acid amide hydrolase in the hypothalamus of rat pups at weaning (21 days old) or adult rats (4 months old). Maternal under-nutrition resulted in a striking decrease in body weight of weaning rats, paralleled by a decrease in the hypothalamic levels of the endocannabinoid anandamide, but not of 2-arachidonoylglycerol. No significant change in the hypothalamic expression of either cannabinoid CB1 receptors or fatty acid amide hydrolase mRNA was detected in any of the three groups of weaned pups. The decrease in pup body weight and hypothalamic anandamide levels was not observable in 4-month-old rats from any of the three groups. These data suggest that maternal under-nutrition causes a decrease in hypothalamic anandamide levels and loss of body weight, and confirm a crucial role for endocannabinoid signalling in neonatal development.

Amidohydrolases↗

Sub-cellular pathology of scrapie: coated pits are increased in PrP codon 136 alanine homozygous scrapie-affected sheep.

Sub-cellular studies of transmissible spongiform encephalopathies (TSEs) have been carried out on several animal species and human beings. However, studies of optimal perfusion-fixed tissues have largely been confined to examination of rodents. Using a recently developed technique, heads of scrapie-affected sheep and controls were perfusion fixed with mixed aldehydes. The obexes were immunohistochemically labelled with PrP antibodies, and the dorsal motor nucleus of the vagal nerve was examined by electron microscopy. Irregular neuritic profiles with highly invaginated membranes, associated with coated pits were found in all scrapie-affected sheep, but not in controls. Interestingly, they were consistently more frequent in the homozygous A(136) sheep. This is the first report describing sub-cellular differences in pathology associated with different PrP genotypes. Rarely, amorphous material, or sparse fibrillar structures, were present in the extracellular space. The changes were often associated with irregular plasmalemma and frequent coated pits. Vacuolation typical of TSEs, dystrophic neurites and variable gliosis were present. Herniation of membranes and organelles from apparently healthy processes into adjacent vacuoles and dendrites was also observed. We suggest that the increase in coated pits and plasmalemma invagination is related to an attempted internalisation of aggregated disease-specific PrP, or protofilaments, from the extracellular space.

ATP-Binding Cassette Transporters↗

Regulation of urine marking in male and female mice: effects of sex steroids.

Effects of sex steroids on urine-marking activity were studied in male, female, and neonatally androgenized female mice. Urine marking was estimated by suspending ceramic tubes that were connected in a horizontal row with a steel rod into the home cage of an isolated mouse. Intact males showed high marking activity, which was diminished after castration. Both testosterone propionate (TP) and estradiol benzoate (EB) were effective in restoring the marking activity of castrated males, while 5-alpha-dihydrotesterone (DHT) did not have any stimulative effects. Intact normal females showed quite low marking activity and ovariectomy further depressed it. TP and DHT enhanced the marking of ovariectomized females, but EB restored the activity only to the preovariectomy level. In intact females which were neonatally androgenized, the marking activity was much higher than that of normal females. The pattern of the change induced by gonadectomy and hormone treatment in these females resembled that in males. Thus, ovariectomy reduced the activity and both TP and EB restored the level. These results indicate that the sexual dimorphism in the urine marking in mice is primarily determined by hormonal environment during early postnatal age. Hormonal control of scent marking is discussed in relation to the studies in other rodents.

Animals↗

Perinatal decortication impairs performance on an 8-arm radial maze task.

Two experiments evaluate the role of the neocortex in rodent spatial learning. In Experiment 1, perinatally decorticated rats and sham-operated controls began ten training sessions at day 200 on an 8-arm radial maze. Decorticated rats made more errors than controls, but showed improvement by the tenth session. In the second experiment, training was extended to determine whether decorticates could eventually match control performance levels if given sufficient training. Spontaneous activity levels were also recorded and compared to maze performance to investigate the relationship between poor performance on the radial maze and activity. More than half of the decorticates reached criterion performance. Decorticates had significantly elevated spontaneous activity levels when compared to controls, and the magnitude of this hyperactivity was related to performance deficits on the radial maze. These results suggest that with extended training decorticates can learn a spatial task. Performance deficits may reflect the hyperactive tendency of decorticates rather than a specific impairment of spatial learning abilities.

Animals↗

Stable expression and secretion of the B-cell epitope of rodent malaria from Mycobacterium bovis BCG and induction of long-lasting humoral response in mouse.

The live bacterial vaccine Mycobacterium bovis BCG (BCG) is a vehicle worth noticing for various protective antigens. The gene encoding the B-cell epitope of the oligopeptide repeating in the circumsporozoite protein (C.S. protein) of the rodent malaria parasite, Plasmodium yoelii, was inserted into the plasmid vector under the control of an expression cassette carrying the promoter and signal sequence of the a antigen derived from Mycobacterium kansasii (k-a). The B-cell epitope was successfully expressed and secreted from BCG as a fusion protein with k-a. This recombinant BCG was administered subcutaneously into BALB/c mice and the antibody production was measured by the enzyme-linked immunosorbent assay (ELISA). Long lasting humoral response was found in one of seven mice.

Animals↗

Long term dietary methoxychlor exposure in rats increases sodium solution consumption but has few effects on other sexually dimorphic behaviors.

Methoxychlor is an insecticide with estrogen-like activity, thus exposure during development might cause sexually dimorphic behavioral alterations. To evaluate this, pregnant rats consumed diets containing 0, 10, 100 or 1000 ppm methoxychlor from gestational day 7, and offspring continued on these diets until postnatal day (PND) 77. Assessments of sexually dimorphic behaviors in offspring indicated that intake of a 3.0% sodium chloride solution was significantly increased (41%) in males and females of the 1000 ppm group. No treatment group differed from controls in open field nor running wheel activity, play behavior, nor 0.3% saccharin solution intake. Offspring of the 1000 ppm group showed significantly decreased body weight, reaching 17% less than controls at PND 77, but not clearly related to their salt solution intake. During pregnancy, 1000 ppm dams consumed 23% less food and weighed 10% less than controls, but this did not affect litter outcomes. These results indicate that in rodents, developmental and chronic exposure to dietary methoxychlor alters the sexually dimorphic behavior of salt-solution intake in young adults of both sexes. Similar behavioral alterations with other xenoestrogens, and the potential for interactions among xenoestrogens, suggest that this report may minimize the true effects of dietary methoxychlor exposure.

Animals↗

How to make a placenta: mechanisms of trophoblast cell differentiation in mice--a review.

The word placenta is derived from the Latin term meaning 'flat cake'. Despite the rather humble name, the placenta is an amazing organ that forms both the interface for selective delivery of nutrients from the mother to the fetus and also re-directs maternal metabolic, endocrine, cardiovascular and immune functions to promote fetal survival and growth. These two functions are fulfilled by different specialized trophoblast cell subtypes, and my laboratory has been studying how their formation and functions are regulated during placental development. Through molecular studies in cultured cells and tissues, genetic studies in mice, and comparative analysis of placentas from humans, rodents and farm animals, it is now possible to describe molecular pathways that control the development of all major trophoblast cell subtypes and structures of the placenta. The work has revealed an intricate complexity of cell-cell interactions, environmental factors, and molecular networks that control normal development.

Animals↗

A longitudinal study of campylobacter infection of broiler flocks in Great Britain.

One hundred flocks associated with five integrated poultry companies were monitored for one production cycle to investigate risk factors for campylobacter infection of poultry broiler flocks. Bacteriological samples were collected from one house of birds on each site at weekly intervals from 3 to 4 weeks of age until the birds were infected with campylobacter or the flock was depopulated (whichever was sooner). Environmental samples were obtained from 20 houses after cleansing and disinfection of the site before chick arrival. Conventional methods were used for the isolation of campylobacter. Questionnaires were used to collect information on potential risk factors for campylobacter infection. Discrete-time survival analysis was used to assess the influence of various exposures on the age at which the flock was infected with campylobacter. More than 40% of flocks were infected with campylobacter by the time the chicks were 4 weeks old and >90% by 7 weeks. Infection spread rapidly to most birds in a flock. Infection was not predictable by campylobacter status of the last flock reared on the site. (However, because most flocks were infected, the power to detect such an association was poor.) There was no evidence of environmental survival of campylobacters in broiler houses after adequate cleansing and disinfection. The most important predictors of protection from campylobacter were related to effective hygiene barriers (such as housing birds in buildings in a good state of repair, appropriate usage of disinfectant boot dips and a high standard of cleansing and disinfection of the drinking-water equipment). There was no evidence that rodents were a source of infection (but most sites operated effective vermin-control programmes).

Animal Husbandry↗

Contrary to other non-photic cues, acute melatonin injection does not induce immediate changes of clock gene mRNA expression in the rat suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) contain the main clock of the mammalian circadian system. The endogenous oscillation machinery involves interactive positive and negative transcriptional and posttranslational feedback loops involving the clock genes Per1, Per2, Per3, Clock, Bmal1, Cry1 and Cry2. The SCN endogenous oscillation is entrained to 24 h by the light/dark cycle. Light induced regulation of Per1 and Per2 mRNA expression have been suggested to take part in the clock resetting. However, other factors have chronobiotic and synchronizing effects on SCN activity. Especially, the nocturnal pineal gland hormone, melatonin, which is involved in the regulation of both circadian and seasonal rhythms, is known to feedback on the SCN. Melatonin applied on SCN slices immediately phase-shifts their neuronal electrical activity, while daily injections of melatonin to free running rodents resynchronize their locomotor activity to 24 h. To determine whether melatonin feedback control on SCN activity implicates transcriptional regulation of the clock genes, we monitored the expression pattern of Per 1, 2, 3, Bmal1, Cry1 and AVP mRNAs after a single melatonin injection at the end of the subjective day. Results showed that melatonin injection affected none of the mRNA expression pattern during the first circadian night. Per1, Per3, Bmal1 and AVP expression patterns were, however, significantly but differentially affected, during the second subjective night after the melatonin injection. The present results strongly suggest that the immediate phase shifting effect of melatonin on the SCN molecular loop implicates rather post-translational than transcriptional mechanisms.

ARNTL Transcription Factors↗

Toxicological evaluation of the staircase test for assessing fine motor movements.

A variety of tests have been developed to study neurotoxicant-related changes in motor function. However, despite recent advances, there remains a need for simple and specific tests of fine motor movements. Accordingly, we chose to evaluate whether a method developed for measuring changes in skilled movements following motor pathway lesions in rodents would provide a sensitive, specific, and economical approach to assessing fine motor control in the toxicology laboratory. We measured skilled paw reaching using the "staircase test" developed by Montoya et al. [Prog. Brain Res. 82 (1990) 459], in which a rat retrieves food pellets by reaching down from a central platform to a series of descending steps on either side, grasping the pellets in its forepaw, and lifting them to its mouth. Staircase boxes were scaled for the body weights of young adult male (350 g) and female (250 g) Long-Evans rats. Studies were conducted using harmaline, a tremorigen; scopolamine; methyl scopolamine; and 2,4-dithiobiuret (DTB), a compound that causes muscle weakness by interfering with cholinergic transmission at the neuromuscular junction. Harmaline (0, 1.0, 3.0, and 10.0 mg/kg) reduced pellet retrieval only at a dose that also caused visible tremor. Both scopolamine (0, 0.1, 0.3, and 1.0 mg/kg) and methyl scopolamine (0, 0.104, 0.312, and 1.04 mg/kg) impaired pellet retrieval; scopolamine was more effective than methyl scopolamine. DTB (5 daily doses of 0, 0.1, 0.2, and 0.5 mg/kg) had no effect on retrieval, even when causing visible signs of weakness. These data cast doubt on the utility of this method for detecting and quantifying subtle chemical-induced changes in motor function in rats.

Analysis of Variance↗

Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonin-deficient C57BL mice and melatonin-proficient C3H mice.

In rodents, the nocturnal rise and fall of arylalkylamine N-acetyltransferase (AANAT) activity controls the rhythmic synthesis of melatonin, the hormone of the pineal gland. This rhythm involves the transcriptional regulation of the AANAT by two norepinephrine (NE)-inducible transcription factors, e.g. the activator pCREB (phosphorylated Ca2+/cAMP-response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). Most inbred mouse strains do not produce melatonin under standard laboratory light/dark conditions. As melatonin-deficient mice are often the founders for transgenic animals used for chronobiological experimentations, molecular components of neuroendocrine signalling in the pineal gland as an integral part of clock entrainment mechanisms have to be deciphered. We therefore compared calcium signalling, transcriptional events and melatonin synthesis in the melatonin-deficient C57BL mouse and the melatonin-proficient C3H mouse. Pineal glands and primary pinealocytes were cultured and stimulated with NE or were collected at various times of the light/dark (LD) cycle. Changes in intracellular calcium concentrations, the phosphorylation of CREB, and ICER protein levels follow similar dynamics in the pineal glands of both mouse strains. pCREB levels are high during the early night and ICER protein shows elevated levels during the late night. In the C57BL pineal gland, a low but significant increase in melatonin synthesis could be observed upon NE stimulation, and, notably, also when animals were exposed to long nights. We conclude that the commonly used C57BL mouse is not completely melatonin-deficient and that this melatonin-deficiency does not affect molecular details involved in regulating transcriptional events of melatonin synthesis.

Adrenergic alpha-Agonists↗

Hypothalamic appetite-regulating neuropeptide mRNA levels in cachectic nude mice bearing human tumor cells.

We previously reported that the human melanoma cell line, SEKI, induces severe weight loss in nude mice. In the present study, we examined the expression of weight-regulating neuropeptide mRNAs in the hypothalamus of this cancer cachectic model by using a sensitive quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method and in situ hybridization. mRNA levels of neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) in the whole hypothalamus were elevated significantly in the SEKI mice as compared with control mice. In situ hybridization showed that NPY and CRH mRNA were upregulated in the arcuate nucleus and the paraventricular nucleus, respectively. There were no significant differences in melanin-concentrating hormone (MCH), orexin (OX), and cholecystokinin mRNA levels between the SEKI and control mice. These results suggest that the NPYergic system is functioning in the rodent model of cancer cachexia; however, the role of the CRHergic system in energy homeostasis remains to be elucidated. This is the first report of the hypothalamic neuropeptide response to cachexia-inducing human cells.

Animals↗

Replication of enhancer-deficient amphotropic murine leukemia virus in human cells.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. The generation of replication-competent virus is considered less likely with vectors that delete the viral transcription elements. This conclusion is based on data obtained in rodents, where MLV replication depends on the expression of viral genes under the control of 75-bp enhancer elements in the long terminal repeat. We demonstrate here that in some human cells replication of amphotropic MLV is possible in the absence of these enhancer elements. Replication of the enhancer-deficient virus MLV-(MOA)Delta E is observed in selected human sarcoma and B lymphoma lines and proceeds at a lower rate than that of the intact virus. No insertion of a foreign promoter or enhancer into the long terminal repeat was detected. Our data suggest the presence of a secondary enhancer element within the MLV provirus that can in selected human cells mediate virus transcription and replication in the absence of the 75-bp U3 enhancers.

3T3 Cells↗

Pescadillo is essential for nucleolar assembly, ribosome biogenesis, and mammalian cell proliferation.

Mutation of the zebrafish pescadillo gene blocks expansion of a number of tissues in the developing embryo, suggesting roles for its gene product in controlling cell proliferation. We report that levels of the pescadillo protein increase in rodent hepatocytes as they enter the cell cycle. Pescadillo protein localizes to distinct substructures of the interphase nucleus including nucleoli, the site of ribosome biogenesis. During mitosis pescadillo closely associates with the periphery of metaphase chromosomes and by late anaphase is associated with nucleolus-derived foci and prenucleolar bodies. Blastomeres in mouse embryos lacking pescadillo arrest at morula stages of development, the nucleoli fail to differentiate and accumulation of ribosomes is inhibited. We propose that in mammalian cells pescadillo is essential for ribosome biogenesis and nucleologenesis and that disruption to its function results in cell cycle arrest.

3T3 Cells↗