Fos expression in the brains of rats performing an attentional set-shifting task.

Impairments in executive function and cognitive control are a common feature of neuropsychiatric and neurodegenerative disorders. A promising behavioral paradigm for elucidating the neural mechanisms of executive function is extradimensional/intradimensional (ED/ID) shifting, which places demands on executive function by requiring the adjustment of behavioral responses based on affective or attentional information. To augment the understanding of the brain systems required for these aspects of executive function, we examined the induction of Fos protein in rats tested in the ED/ID paradigm. We found increased Fos-like immunoreactivity (Fos-LI) in several cortical areas, including medial and orbital frontal cortex (OFC), in rats performing affective or attentional shifts relative to rats performing control discriminations. However, increased Fos-LI was also present in rats that performed a yoked number of additional control discrimination trials, without affective or attentional shifting. These observations suggest that cortical networks required for affective and attentional shifting are also activated during comparable discrimination tasks that do not require shifting, consistent with a role for these networks in monitoring ongoing behavior even in situations in which adaptation to changing behavioral demands is not required.

Factors associated with concentrations of select cytokine and acute phase proteins in dairy cows with naturally occurring clinical mastitis.

The objective of the current observational study was to determine the potential associations between cow factors, clinical mastitis (CM) etiology, and concentrations of select acute phase proteins and cytokines in milk from affected quarters of cows with CM. Cows with CM (n=197) were grouped based on systemic disease severity, milk culture result, parity, days in milk (DIM), previous CM occurrence, and season of the year when CM occurred. Concentrations of lipopolysaccharide-binding protein (LBP), haptoglobin (Hp), BSA, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-8, IL-10, IL-12, transforming growth factor (TGF)-alpha, and TGF-beta and activity of lactate dehydrogenase (LDH) were evaluated. Differences in the least squares means log(10) transformed concentrations of these proteins were compared using multiple linear regression mixed models. The milk concentrations of LBP, Hp, IL-1beta, IL-10, and IL-12, and activity of LDH in milk were higher in cows with moderate to severe versus mild systemic disease. The concentrations of Hp, BSA, IL-1beta, and IL-10 in milk were higher in cows with a gram-negative versus gram-positive milk culture result. Season of the year when CM occurred was associated with the concentration of all proteins evaluated except for IL-1beta and IL-12. Concentrations were higher in the winter versus summer except for Hp and TGF-beta, for which the opposite was true. Concentrations of LBP, IL-10, and IL-12, and LDH activity in milk were associated with DIM group. Except for LBP, these proteins were lower in cows with CM during the first 60 DIM versus those in mid or later lactation. Interferon-gamma, TNF-alpha, and IL-8 were undetectable in 67, 31, and 20% of samples, respectively. Detection of IFN-gamma and IL-8 was associated with season, and detection of TNF-alpha and IL-8 was associated with systemic disease severity. The current study provides the most comprehensive report of milk concentrations of innate immune response proteins in cows with naturally occurring CM and identifies factors that potentially influence those concentrations. Further investigation into the seasonal variation of cytokine production and its potential effect on the outcome of CM is warranted. Furthermore, the results of this study provide useful data for planning future studies examining the role of the innate immune response in CM.

Enhanced host immune recognition of mastitis causing Escherchia coli in CD-14 transgenic mice.

Escherchia coli causes mastitis, an economically significant disease in dairy animals. E. coli endotoxin (lipopolysaccharide, LPS) when bound by host membrane proteins such as CD-14, causes release of proinflammatory cytokines recruiting neutrophils as an early, innate immune response. Excessive proinflammatory cytokines causes tissue damage, compromising mammary function. If present, soluble CD-14 (sCD-14) might out compete membrane bound CD-14, lessening the severity of the inflammatory response. To test this hypothesis transgenic mice, expressing sCD-14 in their milk (31 to 316 microg/ml), were evaluated. A cell culture study demonstrated, in the presence of LPS, milk from transgenic mice increased secretion of cytokine IL-8 compared to milk from nontransgenic littermates (18 +/- 3 vs. 35 +/- 2 ng/mL, p < 0.001). To assess protection afforded by the transgene, glands were infused with E. coli. Recovery of bacteria showed no clear relationship between sCD14 concentration and the number of organisms recovered; however, there was a strong relationship between sCD14 concentration and edema (r(2) = 0.999, p < 0.001), as measured by weight of fluid in harvested glands. Highest expressing lines had the least edema, suggesting the presence of sCD14 had an effect on reducing the inflammatory response to E. coli, thus, possibly protecting against gland tissue damage.

Comparison of Holstein and Jersey innate immune responses to Escherichia coli intramammary infection.

Mastitis is one of the most prevalent diseases in cattle and remains among the most costly diseases to the dairy industry. Various surveys have indicated a greater prevalence of and risk for mastitis in Holstein cows than in Jersey cows. The innate immune system comprises the immediate host defense mechanisms that respond to infection, and differences in the magnitude and rapidity of this response are known to influence susceptibility to and clearance of infectious pathogens. The reported differences in the prevalence of mastitis between Holstein and Jersey cows may suggest the occurrence of breed-dependent differences in the innate immune response to intramammary infection. The objective of the current study was to compare the acute phase and cytokine responses of Holstein and Jersey cows following intramammary infection by the bacterial pathogen Escherichia coli, a leading cause of clinical mastitis. All cows in the study were in similar stages of lactation, of the same parity, subjected to the same housing and management conditions, and experimentally infected on the same day with the same inoculum preparation. Before and after infection, the following innate immune parameters were monitored: bacterial clearance; febrile response; induction of the acute phase proteins serum amyloid A and lipopolysaccharide-binding protein; alterations in total and differential white blood cell counts; changes in milk somatic cell counts and mammary vascular permeability; and induction of the cytokines IFN-gamma, IL-1beta, IL-8, IL-12, and tumor necrosis factor-alpha. Overall innate immune responses were similar between the 2 breeds; however, temporal differences in the onset, cessation, and duration of several responses were detected. Despite these differences, intramammary clearance of E. coli was comparable between the breeds. Together, these data demonstrate a highly conserved innate immune response of Holstein and Jersey cows to E. coli intramammary infection.

Innate immune response to intramammary Mycoplasma bovis infection.

The objective of the current study was to characterize the systemic and local innate immune response of dairy cows to IMI with Mycoplasma bovis, a pathogen of growing concern to the dairy industry. Ten Holstein cows were each infused in 1 quarter with M. bovis and studied for a 10-d period. Acute phase protein synthesis, which reflects 1 parameter of the systemic response to infection, was induced within 108 h of infection, as evidenced by increased circulating concentrations of lipopolysaccharide binding protein and serum amyloid A. Transient neutropenia was observed from 84 to 168 h postinfection, whereas a constant state of lymphopenia and thrombocytopenia was observed from 84 h until the end of the study. Milk somatic cell counts initially increased within 66 h of M. bovis infusion and remained elevated, relative to control (time 0) concentrations, for the remainder of study. Increased milk concentrations of BSA, which reflect increased permeability of the mammary epithelial-endothelial barrier, were evident within 78 h of infection and were sustained from 90 h until the end of the study. Milk concentrations of several cytokines, including IFN-gamma, IL-1beta, IL-10, IL-12, tumor growth factor-alpha, and tumor necrosis factor-alpha, were elevated in response to infection over a period of several days, whereas increases in milk IL-8 were of a more limited duration. Complement activation, reflected by increased milk concentrations of complement factor 5a, was also observed over several days. Despite the indication by these observed changes that the cows mounted a prolonged inflammatory response to M. bovis intramammary infection, all quarters remained infected throughout the study with persistently high concentrations of this bacterium. Thus, a sustained inflammatory response is not sufficient to eradicate M. bovis from the mammary gland and may reflect the ongoing struggle of the host to clear this persistent pathogen.

Effect of mastitis on milk perchlorate concentrations in dairy cows.

Recent surveys have identified the presence of perchlorate, a natural compound and environmental contaminant, in forages and dairy milk. The ingestion of perchlorate is of concern because of its ability to competitively inhibit iodide uptake by the thyroid and to impair synthesis of thyroid hormones. A recent study established that milk perchlorate concentrations in cattle highly correlate with perchlorate intake. However, there is evidence that up to 80% of dietary perchlorate is metabolized in clinically healthy cows, thereby restricting the available transfer of ingested perchlorate into milk. The influence of mastitis on milk perchlorate levels, where there is an increase in mammary vascular permeability and an influx of blood-derived components into milk, remains unknown. The present study examined the effect of experimentally induced mastitis on milk perchlorate levels in cows receiving normal and perchlorate-supplemented diets. Over a 12-d period, cows were ruminally infused with 1 L/d of water or water containing 8 mg of perchlorate. Five days after the initiation of ruminal infusions, experimental mastitis was induced by the intramammary infusion of 100 microg of bacterial lipopolysaccharide (LPS). Contralateral quarters infused with phosphate-buffered saline served as controls. A significant reduction in milk perchlorate concentration was observed in the LPS-challenged glands of animals ruminally infused with either water or perchlorate. In control glands, milk perchlorate concentrations remained constant throughout the study. A strong negative correlation was identified between mammary vascular permeability and milk perchlorate concentrations in LPS-infused glands. These findings, in the context of a recently published study, suggest that an active transport process is operative in the establishment of a perchlorate concentration gradient across the blood-mammary gland interface, and that increases in mammary epithelial and vascular endothelial permeability lead to a net outflow of milk perchlorate. The overall finding that mastitis results in lower milk perchlorate concentrations suggests that changes in udder health do not necessitate increased screening of milk for perchlorate.

Sequencing, chromosomal mapping, and functional characterization of bovine FLICE-like inhibitory protein (FLIP).

FLICE-like inhibitory protein (FLIP) has been shown in both humans and mice to inhibit apoptosis and NF-kappaB activation induced by pro-inflammatory mediators. The activation of NF-kappaB and the induction of apoptosis are critical events in the pathogenesis of a variety of disease states in cattle, including mastitis. Since FLIP is known to moderate these events in other species, we mapped the bovine FLIP gene, sequenced bovine FLIP cDNA, and characterized its expression in cultured primary bovine endothelial cells. Sequencing of bovine FLIP revealed approximately 83, 74, and 68% amino acid sequence identity to its porcine, human, and murine orthologs, respectively. Bovine FLIP was mapped to chromosome 2 by radiation hybrid mapping. Interestingly the region to which bovine FLIP maps contains a putative quantitative trait locus for functional herd life which is an indicator of a cow's ability to survive involuntary culling due primarily to mastitis and infertility. In addition to sequencing and mapping, the function of bovine FLIP was studied. Over-expression of bovine FLIP protected against bacterial lipopolysaccharide (LPS)- and TNF-alpha-induced apoptosis in bovine endothelial cells consistent with previous studies of human FLIP. In addition, elevated expression of bovine FLIP blocked LPS- and TNF-alpha-induced upregulation of NF-kappaB-dependent gene products as assayed by E-selectin expression. Only the full-length bovine FLIP protein could inhibit NF-kappaB activation induced by LPS, whereas the death effector domain region alone was able to inhibit TNF-alpha-induced NF-kappaB activation. Together, these data demonstrate the conservation of FLIP's ability to inhibit apoptosis and to downregulate NF-kappaB activation across species.

Increased milk levels of transforming growth factor-alpha, beta1, and beta2 during Escherichia coli-induced mastitis.

Among the gram-negative bacteria that cause mastitis, Escherichia coli are the most prevalent. The innate immune system provides initial protection against E. coli infection by detecting the presence of the foreign pathogens and by mounting an inflammatory response, the latter of which is mediated by cytokines such as IL-1beta, IL-8, and tumor necrosis factor (TNF)-alpha. Although changes in these cytokines during mastitis have been well-described, it is believed that other mediators moderate mammary gland inflammatory responses as well. The growth factors/cytokines transforming growth factor (TGF)-alpha, TGF-beta1, and TGF-beta2 are all expressed in the mammary gland and have been implicated in regulating mammary gland development. In other tissues, these growth factors/cytokines have been shown to moderate inflammation. The objective of the current study was to determine whether TGF-alpha, TGF-beta1, and TGF-beta2 milk concentrations were altered during the course of E. coli-induced mastitis. The contralateral quarters of 11 midlactating Holstein cows were challenged with either saline or 72 cfu of E. coli, and milk samples were collected. Basal milk levels of TGF-alpha, TGF-beta1, and TGF-beta2 were 98.81 +/- 22.69 pg/mL, 3.35 +/- 0.49 ng/mL, and 22.36 +/- 3.78 ng/mL, respectively. Analysis of whey samples derived from E. coli-infected quarters revealed an increase in milk levels of TGF-alpha within 16 h of challenge, and these increases persisted for an additional 56 h. Elevated TGF-beta1 and TGF-beta2 milk concentrations were detected in E. coli-infected quarters 32 h after challenge, and these elevations were sustained throughout the study. Because TGF-alpha, TGF-beta1, and TGF-beta2 have been implicated in mediating inflammatory processes, their induction during mastitis is consistent with a role for these molecules in mediating mammary gland host innate immune responses to infection.

Characterization of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae.

Gram-negative bacteria are responsible for almost one-half of the clinical cases of mastitis that occur annually. Of those gram-negative bacteria that induce mastitis, Klebsiella pneumoniae remains one of the most prevalent. Detection of infectious pathogens and the induction of a proinflammatory response are critical components of host innate immunity. The objective of the current study was to characterize several elements of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae. The inflammatory cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), 2 proteins that contribute to host recognition of gram-negative bacteria, were studied. The contralateral quarters of 7 late-lactating Holstein cows were challenged with either saline or K. pneumoniae, and milk and blood samples were collected. Initial increases in the chemoattractants C5a and IL-8, as well as TNF-alpha, were evident in infected quarters within 16 h of challenge and were temporally coincident with increases in milk somatic cells. Augmented levels of TNF-alpha and IL-8 were observed in infected quarters until >48 h postchallenge, respectively. Elevated levels of IL-12, IFN-gamma, and the antiinflammatory cytokine, IL-10, which were first detected between 12 and 20 h postinfection, persisted in infected quarters throughout the study (>96 h). Initial increases in milk LBP and sCD14 were detected 16 and 20 h, respectively, after challenge. Together, these data demonstrate that intramammary infection with K. pneumoniae elicits a host response characterized by the induction of proinflammatory cytokines and elevation of accessory molecules involved in LPS recognition.

GluR-A-Deficient mice display normal acquisition of a hippocampus-dependent spatial reference memory task but are impaired during spatial reversal.

Acquisition and reversal of a spatial discrimination were assessed in an appetitive, elevated plus-maze task in 4 groups of mice: knockout mice lacking the AMPA receptor subunit GluR-A (GluR1), wild-type controls, mice with cytotoxic hippocampal lesions, and controls that had undergone sham surgery. In agreement with previous studies using tasks such as the water maze, GluR-A(-/-) mice were unimpaired during acquisition of the spatial discrimination task, whereas performance in the hippocampalgroup remained at chance levels. In contrast to their performance during acquisition, the GluR-A(-/-) mice displayed a mild deficit during reversal of the spatial discrimination and were profoundly impaired during discrete trial, rewarded-alternation testing on the elevated T maze. The latter result suggests a short-term, flexible spatial working memory impairment in GluR-A(-/-) mice, which might also underlie their mild deficit during spatial reversal.

Double dissociation of function within the hippocampus: spatial memory and hyponeophagia.

Complete and dorsal hippocampal lesions impaired spatial performance on 2 working memory tasks: rewarded alternation on the T maze and matching to position in the water maze. In contrast, ventral hippocampal lesions had no effect on these tasks, even when task difficulty was increased by the introduction of delays. Ventral lesions did resemble complete lesions in reducing anxiety in 3 commonly used tests of anxiety (social interaction, plus-maze, and hyponeophagia). Dorsal lesions also appeared to be anxiolytic in the social interaction and plus-maze tests, but they did not affect hyponeophagia. Complete- and dorsal-lesioned rats displayed hyperactivity, whereas ventral-lesioned rats did not. These results show a double dissociation between dorsal and ventral hippocampal lesions (hyponeophagia vs. spatial memory), suggesting differentiation of function along the septotemporal axis of this structure.

A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis.

Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.

The time course of the hyperactivity that follows lesions or temporary inactivation of the fimbria-fornix.

Lesions of the hippocampus or the fimbria-fornix produce a pronounced hyperactivity in rats. This effect is thought to be due to the loss of glutamatergic hippocampal inputs to the nucleus accumbens, although the mechanisms involved remain unclear. It has been suggested that the hyperactivity is due to changes in accumbens dopamine receptors, possibly involving the gradual development of denervation supersensitivity. Consistent with this possibility, the present study found that fimbria-fornix transection produced hyperactivity which, although undetectable immediately after surgery, gradually became apparent and then continued to increase over the course of several days. This does not, however, preclude the possibility that there is an immediate increase in activity which is masked by the after effects of surgery. To address this issue, local anaesthetic was infused into the fimbria-fornix via chronic indwelling cannulae, in order to silence the hippocampal inputs to the nucleus accumbens. This procedure impaired spatial working memory on the elevated T-maze and resulted in immediate hyperactivity, suggesting that there may be at least two components to fornix lesion-induced hyperactivity, and that the immediate effects of mechanical fornix lesions on activity levels may be masked by the after effects of surgery per se.

Exposure to febrile temperature modifies endothelial cell response to tumor necrosis factor-alpha.

Fever is an important regulator of inflammation that modifies expression and bioactivity of cytokines, including tumor necrosis factor (TNF)-alpha. Pulmonary vascular endothelium is an important target of TNF-alpha during the systemic inflammatory response. In this study, we analyzed the effect of a febrile range temperature (39.5 degrees C) on TNF-alpha-stimulated changes in endothelial barrier function, capacity for neutrophil binding and transendothelial migration (TEM), and cytokine secretion in human pulmonary artery endothelial cells (EC). Permeability for [(14)C]BSA tracer was increased by treatment with TNF-alpha, and this effect was augmented by incubating EC at 39.5 degrees C. Treating EC with 2. 5 U/ml TNF-alpha stimulated an increase in subsequent neutrophil adherence and TEM. Incubating EC at 39.5 degrees C caused a 30% increase in TEM but did not modify the enhancement of neutrophil adherence or TEM by TNF-alpha treatment. Analysis of cytokine expression in EC cultures exposed to TNF-alpha at either 37 degrees or 39.5 degrees C revealed three patterns of temperature and TNF-alpha responsiveness. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-8 were not detectable in untreated EC but were increased after TNF-alpha exposure, and this increase was enhanced at 39.5 degrees C. IL-6 expression was also increased with TNF-alpha exposure, but IL-6 expression was lower in 39.5 degrees C EC cultures. Transforming growth factor-beta(1) was constitutively expressed, and its expression was not influenced either by TNF-alpha or exposure to 39.5 degrees C. These data demonstrate that clinically relevant shifts in body temperature might cause important changes in the effects of proinflammatory cytokines on the endothelium.

Antiplatelet effects of R,S-(meso)-alpha, alpha'-bis[3-(N,N-diethylcarbamoyl) piperidino]-p-xylene ex vivo in the dog and in vivo in the mouse.

1. The nipecotamide alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene (A-1) is a platelet aggregation inhibitor. The meso diastereomer A-1c is superior in potency and duration to the synthetic diastereomeric mixture consisting of the R,R-, S,S-, and R,S- (meso) isomers in inhibiting collagen-induced platelet aggregation ex vivo in the dog. 2. A-1c also is more potent and longer acting than A-1 in protecting mice from collagen+epinephrine-induced thromboembolic death. 3. The mechanism of antiplatelet action of this compound appears to be related to its ability to prevent agonist-induced inhibition of platelet cyclic adenosine monophosphate (cAMP) levels.