Atypical play experiences in the juvenile period has an impact on the development of the medial prefrontal cortex in both male and female rats.

In rats reared without play, or with limited access to play during the juvenile period, the dendrites of pyramidal neurons of the medial prefrontal cortex (mPFC) exhibit more branching than rats reared with more typical levels of play. This suggests that play is critical for pruning the dendritic arbor of these neurons. However, the rearing paradigms typically used to limit play involve physical separation from a peer or sharing a cage with an adult, causing stress that may disrupt pruning. To limit this potentially confounding source of stress, we used an alternative approach in this study: pairing playful Long Evans rats (LE) with low playing Fischer 344 (F344) rats throughout the juvenile period. We then examined the morphology of medial prefrontal cortex (mPFC) neurons, predicting that pruning should be reduced. LE rats reared with another LE rat had significantly greater pruning of mPFC pyramidal neurons compared to LE rats reared with a F344 partner. Furthermore, in previous studies, only one sex or the other was used, whereas in the present rearing paradigm, both sexes were tested, showing that play influences neuronal pruning in both. The neurons of the play deficient LE rats not only occupied more space, as determined by convex hull analyses, but the dendrites were also longer than in rats with more typical play experiences. Unlike studies using more stressful rearing paradigms, the present effects were limited to the apical dendritic projections, suggesting that the previously reported effects on the basilar dendrites may have resulted from developmental disruptions caused by stress. If correct, the present findings indicate that play experienced over the juvenile period affects how mPFC neurons develop and function.

Seasonal differences in the morphology and spine density of hippocampal neurons in wild ground squirrels.

Seasonally reproducing small mammals often undergo changes in brain anatomy throughout the year. Much of the research on this seasonal neuroplasticity has focused on changes in hippocampus volume and neurogenesis, with relatively little attention paid to neuronal morphology. Here, we test for sex, season and sex-season interaction effects on hippocampal neuron morphology and dendritic spine density in a seasonally reproducing rodent: Richardson's ground squirrel (Urocitellus richardsonii). We quantified the morphology and spine densities of Golgi-stained pyramidal neurons and granule cells in the hippocampus and tested for differences between sexes and seasons with generalized linear models. Although we found no significant sex differences or sex-season interaction effects on any of our morphological measurements, there were significant differences in neuron morphology and spine density between breeding and non-breeding seasons. In the non-breeding season, ground squirrels had CA1 neurons with longer basal dendrites with more branches than in the breeding season. Non-breeding season animals also had higher apical and basal dendrite spine density in CA1 and CA3 neurons than breeding-season animals. Conversely, the spine densities of CA1 somata and granule cells were higher in breeding than in non-breeding season. These differences in neuron morphology and spine density between breeding and non-breeding seasons likely arise from a combination of activity levels, stress hormones, and photoperiod. Although the functional implications of seasonal changes in hippocampal neuron morphology and spine density are uncertain, our data suggest that ground squirrels may be a good model for understanding seasonal neuroplasticity in mammals.

Metastable dynamics of neural circuits and networks.

Cortical neurons emit seemingly erratic trains of action potentials or "spikes," and neural network dynamics emerge from the coordinated spiking activity within neural circuits. These rich dynamics manifest themselves in a variety of patterns, which emerge spontaneously or in response to incoming activity produced by sensory inputs. In this Review, we focus on neural dynamics that is best understood as a sequence of repeated activations of a number of discrete hidden states. These transiently occupied states are termed "metastable" and have been linked to important sensory and cognitive functions. In the rodent gustatory cortex, for instance, metastable dynamics have been associated with stimulus coding, with states of expectation, and with decision making. In frontal, parietal, and motor areas of macaques, metastable activity has been related to behavioral performance, choice behavior, task difficulty, and attention. In this article, we review the experimental evidence for neural metastable dynamics together with theoretical approaches to the study of metastable activity in neural circuits. These approaches include (i) a theoretical framework based on non-equilibrium statistical physics for network dynamics; (ii) statistical approaches to extract information about metastable states from a variety of neural signals; and (iii) recent neural network approaches, informed by experimental results, to model the emergence of metastable dynamics. By discussing these topics, we aim to provide a cohesive view of how transitions between different states of activity may provide the neural underpinnings for essential functions such as perception, memory, expectation, or decision making, and more generally, how the study of metastable neural activity may advance our understanding of neural circuit function in health and disease.

Stimulus-specific assembly of enhancer complexes on the tumor necrosis factor alpha gene promoter.

The human tumor necrosis factor alpha (TNF-alpha) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-alpha enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-alpha gene transcription and selectively associates with the TNF-alpha promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-alpha transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.

Detection of intracellular interferon-gamma by light microscopy using an immunoperoxidase technique: correlation with the corresponding mRNA and protein product.

Identifying individual cytokine-producing cells may help to acquire insight into immunological processes. This study was designed to adapt a technique for the detection of individual cytokine-producing cells from an immunofluorescence to an immunoperoxidase staining procedure. The production of interferon-gamma (IFN-gamma) by anti-CD3-activated cloned human T cells was used as a model system. After the conditions for the staining procedure were optimized, the immunoperoxidase technique was slightly more sensitive than the immunofluorescence technique. The intracellular staining for IFN-gamma was preceded or paralleled by IFN-gamma mRNA production and followed by accumulation of IFN-gamma in the supernatant. It is concluded that intracellular IFN-gamma can easily be detected using an immunoperoxidase procedure. This procedure is highly sensitive and allows quantification of the production of multiple cytokines by counting the percentage of positively staining cells.

TNF-alpha promoter polymorphisms, production and susceptibility to multiple sclerosis in different groups of patients.

TNF-alpha production in whole blood cultures upon stimulation with LPS was determined in 179 individuals from 61 families in order to characterise the magnitude of inherited differences in TNF-alpha production. The three families characterised by highest TNF production showed 7.1 +/- 0.3 ng TNF/ml upon culture with 10 ng LPS and 10.2 +/- 0.2 ng TNF/ml upon culture with 1000 ng LPS. in contrast to the three families characterised by the lowest TNF production that showed a production of 1.6 +/- 0.1 ng TNF upon culture with 10 ng and 2.5 +/- 0.2 ng/ml upon culture with 1000 ng LPS/ml. This difference could not be attributed to the promoter polymorphisms -308 G to A. -238 G to A or -376 G to A, although the -238 GA donors produced 2.1 +/- 0.9 ng TNF upon culture with 10 ng endotoxin compared to 3.2 +/- 2.2 ng TNF for the -238 GG donors. In line with these results the frequency of the -238 GG genotype was increased in hospitalized MS patients in a nursing home (100% 238GG, n = 57) compared to MS patients in an outpatient's clinic (94% 238GG, n = 98) or Dutch controls (90% 238GG, n = 180). These results suggest that the -238 GG genotype is differently distributed in hospitalized MS patients in a nursing home.

Polymorphism within the tumor necrosis factor alpha (TNF) promoter region in patients with ankylosing spondylitis.

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the location of the TNF gene in the vicinity of the HLA-B locus, and the prominent role in inflammation of its product, we investigated the association between AS and two G to A transition polymorphisms located at position -238 and -376 in the promoter region of the TNF gene. The distribution of the TNF alleles was determined in 86 HLA-B27+ AS patients and 163 healthy controls. From the 86 AS patients, 33 suffered from acute anterior uveitis (AAU). No significant difference for the TNF-376 polymorphism in AS and healthy controls was observed. The frequency of the TNF-238A allele in HLA-B27+ AS patients was significantly decreased compared to random controls (p = 0.021). However, the frequency of the TNF-238A allele in HLA-B27+ AS patients was not significantly different from that observed in HLA-B27+ healthy individuals (p = 0.6). Assessment of association showed that the TNF-238G allele is in linkage disequilibrium with the HLA-B27 allele (delta = 0.053; P = 0.008). Therefore, we conclude that the association between TNF-238G and AS is secondary to the HLA-B27 gene and that TNF-238 and-TNF-376 alleles are not likely to be involved in the susceptibility to AS.

Tumor necrosis factor alpha-308 gene variants in relation to major histocompatibility complex alleles and Felty's syndrome.

The location of the human TNF genes within the MHC complex has prompted much speculation about the role of TNF alleles in the etiology of MHC-associated autoimmune diseases. On sequencing the 5' regulatory region of the human TNFA gene a G (TNFA-308G) to A (TNFA-308A) transition polymorphism at position -308 was discovered. We have developed a simple PCR assay to facilitate the screening of the -308 polymorphism at the DNA level. In view of the possible linkage between the TNFA-308A allele and a certain MHC type, TNFA-308 genotypes in HLA-typed healthy individuals (n = 88) were determined. A statistically significant association between the TNFA-308A allele and HLA-DR3, DQB1*0201, DQA1*0501, A1, B8, and the NcoI 5.5-kb RFLP of the TNFB gene was observed. In addition, we determined the frequency of the TNFA-308A allele in patients with FS (n = 13), an HLA-DR4-associated disease. In this study, no association was found of Felty's syndrome with the TNFA-308A allele, indicating that this allele does not appear to be a susceptibility factor for FS.

Qualitative gene profiling: a novel tool in genomics and in pharmacogenomics that deciphers messenger RNA isoforms diversity.

RNA splicing is a tightly regulated process. It is essential for gene expression and, therefore, intervenes in every biological phenomenon in mammals. RNA splicing regulation is cell type-specific in such a way that a cellular situation can be characterised by its repertoire of spliced events, the spliceome. Comparison of the splicing repertoire of two situations identifies alternative exons and introns. This regulation involves cis-acting sequences and transacting factors. Mutations, as well as modifications of signalling pathways, can alter the accuracy of splicing. Since deletion of exons or retention of introns within coding sequences modifies the corresponding proteins and functional domains of proteins are encoded by contiguous exons, identifying changes in the spliceome pinpoints functional domains, which are specifically regulated at the level of RNA splicing. We have developed a new method of gene profiling, qualitative gene profiling, that allows the comparative study of the repertoires of spliced events that characterise distinct physiopathological situations. We present in this review the different uses of this new genomic technique that can help each step of the R&D process in the pharmaceutical industry, and that represents a short cut towards functional genomics and pharmacogenomics.

Relevance of the tumor necrosis factor alpha (TNF alpha) -308 promoter polymorphism in TNF alpha gene regulation.

Tumor necrosis factor alpha (TNF alpha) is a central mediator of the immunological response and the location of the gene within the major histocompatibility complex (MHC) has prompted much speculation about the role of TNF alpha alleles in inflammatory and MHC-associated autoimmune diseases. A G to A transition polymorphism at position -308 of the TNF alpha promoter/enhancer region has been described. The uncommon -308A allele was shown to be strongly associated with human leukocyte antigen (HLA)-DR3, known to be related to a TNF alpha "high producer" phenotype. In support for a clinical relevance, the -308A allele is implicated in susceptibility for cerebral malaria. In this study, we determined the junctional consequences of the TNF -308 polymorphism. Therefore, we analyzed both allelic forms (TNF alpha(-308G) and TNF alpha(-3O8A)) of the TNF alpha enhancer/promoter region (-598/+108) in a transient transfection system, using chloramphenicol acetyltransferase (CAT) as reporter gene. The T cell line Jurkat and the B cell line Raji served as hosts in these experiments. The results showed no differences in the level of inducible reporter gene expression between the TNF(-3O8G)/CAT and the TNF(-308A)/CAT constructs. These data were confirmed by allele specific TNF alpha transcript quantification (ASTQ) analysis, which demonstrated that both TNF alleles contribute equally to the total amount of mRNA in peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/anti-CD3. In analogy, no difference between the level of transcription of the -308A and -308G alleles was observed in lipopolysaccharide (LPS)-stimulated peripheral blood monocytes. This study indicates that the TNF alpha -308 G to A transition is not responsible for differential TNF alpha production induced by standard in vitro stimuli.

Tumor necrosis factor in Plasmodium falciparum malaria: high plasma level is associated with fever, but high production capacity is associated with rapid fever clearance.

In the present study, we investigated 91 patients with Plasmodium falciparum malaria of different severity in a highly endemic area. Patients were examined at least twice daily until clearance of parasites and fever. Plasma cytokine concentrations without and after ex vivo PHA stimulation of whole blood were determined. On admission we found elevated plasma concentrations of TNF, IFN-gamma, and IL-10 compared to levels during and after chemotherapy. Plasma TNF levels on admission were significantly different between patients with severe and mild malaria (differentiated in schoolchildren and adults). The PHA elicited TNF production capacity of peripheral blood leucocytes was suppressed during the acute phase of malaria. High TNF production capacity was associated with faster fever clearance and parasite clearance and, in patients with severe malaria, with higher blood glucose levels. In conclusion we observed circulating TNF concentrations in malaria patients dependent on the severity of disease, which is itself dependent on age, and an association of a high TNF production capacity with parameters for accelerated cure and good prognosis.

Caspase deficiency alters the murine gut microbiome.

Caspases are aspartate-specific cysteine proteases that have an essential role in apoptosis and inflammation, and contribute to the maintenance of homeostasis in the intestine. These facts, together with the knowledge that caspases are implicated in host-microbe crosstalk, prompted us to investigate the effect of caspase (Casp)1, -3 and -7 deficiency on the composition of the murine gut microbiota. We observed significant changes in the abundance of the Firmicutes and Bacteroidetes phyla, in particular the Lachnospiraceae, Porphyromonodaceae and Prevotellacea families, when comparing Casp-1, -7 and -3 knockout mice with wild-type mice. Our data point toward an intricate relationship between these caspases and the composition of the murine gut microflora.

[Statistical evaluation of residue data for the assessment of predicted values (half-life, withdrawal time) as an example of toltrazuril and enrofloxacin in trout]. / Statistische Auswertung von Rückstandsdaten zur Absicherung von Vorhersagewerten (Halbwertzeit, Wartezeit) am Beispiel von Toltrazuril- und Enrofloxacin-Rückständen in Forellen.

The half-lives and withdrawal times of the veterinary drugs Toltrazuril and Enrofloxacin in trout have been assessed by statistical analysis. Confidence intervals were computed using a normal distribution of residual data and an empirical distribution by the Bootstrap method. Both methods produced similar statistics for the two drugs. Simulation of the residue data according to the regression lines of the decay curves has shown that the Bootstrap method is better for use when the residue patterns are not distributed normally. Using confidence intervals, a statistical mean of withdrawal times can be assessed. Taking into account the decays of the individual antibiotics in all treated trout, tolerance intervals for the regression lines are obtained: the calculated 10-20% longer withdrawal time includes values for which the antibiotic concentration in 95% of the treated trout is decreased below the tolerance level of Toltrazuril or Enrofloxacin.

Allele-specific quantification of TNFA transcripts in rheumatoid arthritis.

Clinical and laboratory studies have suggested a pivotal role for tumor necrosis factor alpha (TNFA) in the pathogenesis of rheumatoid arthritis (RA). Interindividual variation in the expression of TNFA indicates the existence of functionally distinct TNFA alleles that could play a role in susceptibility to TNFA-associated diseases such as RA. In order to determine whether differential TNFA gene expression is present in RA, we studied the relative contribution of TNFA alleles to the total amount of steady-state mRNA in peripheral blood mononuclear cells of RA patients and healthy individuals. Moreover, allelic TNFA mRNA expression was analyzed in synovial biopsy material of RA patients. For this purpose, we used the recently identified C-insertion polymorphism located in the 5' untranslated region of the first exon. The location of this polymorphism within a part of the gene that is transcribed into mRNA allowed us to discriminate between the contribution of each allele to the total amount of TNFA mRNA in heterozygous individuals. The results of this study do not indicate the existence of variation at the level of mRNA transcribed from each TNFA allele by in vitro and physiological stimulation conditions in RA patients. Therefore, our data do not suggest a role for transcriptionally distinct TNFA alleles in the susceptibility to RA.

Polymorphisms within the human tumor necrosis factor-alpha promoter region in human immunodeficiency virus type 1-seropositive persons.

The question is addressed whether particular tumor necrosis factor-alpha (TNF-alpha) polymorphisms are associated with clinical course and outcome of human immunodeficiency virus type 1 (HIV-1) infection. The distribution of four TNF-alpha guanine (G) to adenosine (A) transition polymorphisms at positions -376, -308, -238, and -163 of the 5' promoter region of the TNF-alpha gene was studied in a nested case-control study among HIV-1-seropositive participants of the Amsterdam Cohort. None of the polymorphisms was significantly associated with long-term asymptomatic survival after HIV-1 infection compared with progression to clinical AIDS. Moreover, specific AIDS-defining illnesses or biologic phenotype of the HIV-1 virus were not associated with TNF-alpha alleles. The results of this study do not point toward a role for known TNF-alpha G to A transition polymorphisms in the clinical course of HIV-1 infection.

Detection of a C-insertion polymorphism within the human tumor necrosis factor alpha (TNFA) gene.

We have identified a C-insertion polymorphism in the 5'UTR of the first exon of the human tumor necrosis factor alpha (TNFA) gene. TNFA is a cytokine that plays an important role in the inflammatory response.

Polymorphism of tumour necrosis factor-alpha (TNF-alpha) at position -308 in relation to ankylosing spondylitis.

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the localization, in the proximity of the HLA-B locus, and the biological activities of TNF-alpha, we investigated the association between AS and a single base polymorphism located at position -308 of the TNF-alpha gene. An allele-specific polymerase chain reaction was developed to monitor this polymorphism. The frequency of the TNF-alpha alleles was determined in 66 AS patients and 37 healthy controls. The TNF-alpha allele frequency was not significantly different between AS patients and controls.

Engagement of tumor necrosis factor (TNF) receptor 1 leads to ATF-2- and p38 mitogen-activated protein kinase-dependent TNF-alpha gene expression.

Engagement of the tumor necrosis factor-alpha (TNF-alpha) receptors by the TNF-alpha ligand results in the rapid induction of TNF-alpha gene expression. The study presented here shows that autoregulation of TNF-alpha gene transcription by selective signaling through tumor necrosis factor receptor 1 (TNFR1) requires p38 mitogen-activated protein (MAP) kinase activity and the binding of the transcription factors ATF-2 and Jun to the TNF-alpha cAMP-response element (CRE) promoter element. Consistent with these findings, TNFR1 engagement results in increased p38 MAP kinase activity and p38-dependent phosphorylation of ATF-2. Furthermore, overexpression of MADD (MAP kinase-activating death domain protein), an adapter protein that binds to the death domain of TNFR1 and activates MAP kinase cascades, results in CRE-dependent induction of TNF-alpha gene expression. Thus, the TNF-alpha CRE site is the target of TNFR1 stimulation and mediates the autoregulation of TNF-alpha gene transcription.

A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor alpha gene expression.

The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor alpha (TNF-alpha) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF-alpha gene induction by TCR activation is inhibited, whereas virus induction of the TNF-alpha gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-alpha gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

The tetracycline derivative minocycline differentially affects cytokine production by monocytes and T lymphocytes.

Minocycline is a tetracycline derivative that has beneficial effects in noninfectious forms of arthritis and dermatitis. To investigate whether this effect may be attributed to interference with cytokine production, we studied the effect of minocycline on cytokine production by T cells and monocytes. Minocycline exerted an inhibitory effect on tumor necrosis factor alpha (TNF-alpha) and gamma interferon production by stimulated T cells, whereas the production of interleukin 6 (IL-6) remained unaffected. The effect of minocycline on TNF-alpha mRNA synthesis by T cells was shown to be stimulus specific. T cells stimulated by a Ca2+-independent mode exhibited a decrease in TNF-alpha mRNA in the presence of minocycline, whereas the TNF-alpha mRNA level remained unaffected by minocycline when cells were stimulated in a Ca2+-dependent manner. In contrast to the effect on T cells, addition of minocycline to lipopolysaccharide-stimulated monocytes led to a dose-dependent increase in TNF-alpha and IL-6 production which was paralleled by an enhancement of TNF-alpha mRNA synthesis. These results indicate that minocycline exerts differential effects on the regulation of cytokine production by T cells and monocytes that are partly reflected at the mRNA level. Given the pleiotropic effects of minocycline, it is suggested that the immunostimulatory effect on monocytes might counteract its beneficial properties in the treatment of several forms of chronic inflammation.