Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals : consequences for occupational and environmental medicine

The importance of the isoform CYP2E1 of the human cytochrome P-450 superfamily of enzymes for occupational and environmental medicine is derived from its unique substrate spectrum that includes a number of highly important high-production chemicals, such as aliphatic and aromatic hydrocarbons, solvents and industrial monomers (i.a. alkanes, alkenes, aromatic and halogenated hydrocarbons). Many polymorphic genes, such as CYP2E1, show considerable differences in allelic distribution between different human populations. The polymorphic nature of the human CYP2E1 gene is significant for inter-individual differences in toxicity of its substrates. Since the substrate spectrum of CYP2E1 includes many compounds of basic relevance to industrial toxicology, a rationale for metabolic interactions of different CYP2E1 substrates is provided. In-depth research into the inter-individual phenotypic differences of human CYP2E1 enzyme activities was enabled by the recognition that the 6-hydroxylation of the drug chlorzoxazone is mediated by CYP2E1. Studies on CYP2E1 phenotyping have pointed to inter-individual variations in enzyme activities. There are consistent ethnic differences in CYP2E1 enzyme expression, mostly demonstrated between European and Japanese populations, which point to a major impact of genetic factors. The most frequently studied genetic polymorphisms are the restriction fragment length polymorphisms PstI/RsaI (mutant allele: CYP2E1*5B) located in the 5′-flanking region of the gene, as well as the DraI polymorphism (mutant allele: CYP2E1*6) located in intron 6. These polymorphisms are partly related, as they form the common allele designated CYP2E1*5A. Striking inter-ethnic differences between Europeans and Asians appear with respect to the frequencies of the CYP2E1*5A allele (only approximately 5% of Europeans are heterozygous, but 37% of Asians are, whilst 6% of Asians are homozygous). Available studies indicate a wide variation in human CYP2E1 expression, which are very likely based on complex gene-environment interactions. Major inter-ethnic differences are apparent on the genotyping and the phenotyping levels. Selected cases are presented where inter-ethnic variations of CYP2E1 may provide likely explanations for unexplained findings concerning industrial chemicals that are CYP2E1 substrates. Possible consequences of differential inter-individual and inter-ethnic susceptibilities are related to individual expressions of clinical symptoms of chemical toxicity, to results of biological monitoring of exposed workers, and to the interpretation of results of epidemiological or molecular-epidemiological studies.

The effect of sleep deprivation on BOLD activity elicited by a divided attention task

Sleep loss, widespread in today’s society and associated with a number of clinical conditions, has a detrimental effect on a variety of cognitive domains including attention. This study examined the sequelae of sleep deprivation upon BOLD fMRI activation during divided attention. Twelve healthy males completed two randomized sessions; one after 27 h of sleep deprivation and one after a normal night of sleep. During each session, BOLD fMRI was measured while subjects completed a cross-modal divided attention task (visual and auditory). After normal sleep, increased BOLD activation was observed bilaterally in the superior frontal gyrus and the inferior parietal lobe during divided attention performance. Subjects reported feeling significantly more sleepy in the sleep deprivation session, and there was a trend towards poorer divided attention task performance. Sleep deprivation led to a down regulation of activation in the left superior frontal gyrus, possibly reflecting an attenuation of top-down control mechanisms on the attentional system. These findings have implications for understanding the neural correlates of divided attention and the neurofunctional changes that occur in individuals who are sleep deprived.

Memory and Epileptogenesis in Complex Biological and Simulated Systems

Oscillations of neural activity may bind widespread cortical areas into a neural representation that encodes disparate aspects of an event. In order to test this theory we have turned to data collected from complex partial epilepsy (CPE) patients with chronically implanted depth electrodes. Data from regions critical to word and face information processing was analyzed using spectral coherence measurements. Similar analyses of intracranial EEG (iEEG) during seizure episodes display HippoCampal Formation (HCF)—NeoCortical (NC) spectral coherence patterns that are characteristic of specific seizure stages (Klopp et al. 1996). We are now building a computational memory model to examine whether spatio-temporal patterns of human iEEG spectral coherence emerge in a computer simulation of HCF cellular distribution, membrane physiology and synaptic connectivity. Once the model is reasonably scaled it will be used as a tool to explore neural parameters that are critical to memory formation and epileptogenesis.

Tropomyosin Tm5NM1 spatially restricts src kinase activity through perturbation of rab11 vesicle trafficking

In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules.

Mice behaviorally selected for high Pavlovian fear memory exhibit fear generalization, HPA-axis alterations and increased basal activity in cortico-limbic circuits

Biological factors underlying individual variability in fearfulness and anxiety have important implications for stress-related psychiatric illness including PTSD and major depression. Using an advanced intercross line (AIL) derived from C57BL/6 and DBA/2J mouse strains and behavioral selection over 3 generations, we established two lines exhibiting High or Low fear behavior after fear conditioning. Across the selection generations, the two lines showed clear differences in training and tests for contextual and conditioned fear. Before fear conditioning training, there were no differences between lines in baseline freezing to a novel context. However, after fear conditioning High line mice demonstrated pronounced freezing in a new context suggestive of poor context discrimination. Fear generalization was not restricted to contextual fear. High fear mice froze to a novel acoustic stimulus while freezing in the Low line did not increase over baseline. Enhanced fear learning and generalization are consistent with transgenic and pharmacological disruption of the hypothalamic-pituitary-adrenal axis (HPA-axis) (Brinks, 2009, Thompson, 2004, Kaouane, 2012). To determine whether there were differences in HPA-axis regulation between the lines, morning urine samples were collected to measure basal corticosterone. Levels of secreted corticosterone in the circadian trough were analyzed by corticosterone ELISA. High fear mice were found to have higher basal corticosterone levels than low line animals. Examination of hormonal stress response components by qPCR revealed increased expression of CRH mRNA and decreased mRNA for MR and CRHR1 in hypothalamus of high fear mice. These alterations may contribute to both the behavioral phenotype and higher basal corticosterone in High fear mice. To determine basal brain activity in vivo in High and Low fear mice we used manganese-enhanced magnetic resonance imaging (MEMRI). Analysis revealed a pattern of basal brain activity made up of amygdala, cortical and hippocampal circuits that was elevated in the High line. Ongoing studies also seek to determine the relative balance of excitatory and inhibitory tone in the amygdala and hippocampus and the neuronal structure of its neurons. While these heterogeneous lines are selected on fear memory expression, HPA-axis alterations and differences in hippocampal activity segregate with the behavioral phenotypes. These differences are detectable in a basal state strongly suggesting these are biological traits underlying the behavioral phenotype (Johnson et al, 2011).