Selective contributions of neuronal and astroglial interleukin-1 receptor 1 to the regulation of sleep.

Interactions between sleep and immune function are bidirectional. Although the mechanisms that govern these interactions are not fully elucidated, the pro-inflammatory cytokine, interleukin-1ß (IL-1), is a known regulator of sleep and mediator of immune responses. To further clarify the underlying substrates of sleep and immune interactions, we engineered two transgenic mouse lines that express interleukin-1 receptor 1 (IL1R1) only in the central nervous system (CNS) and selectively on neurons (NSE-IL1R1) or astrocytes (GFAP-IL1R1). During spontaneous sleep, compared to wild type (WT) animals, NSE-IL1R1 and GFAP-IL1R1 mice have more rapid eye movement sleep (REMS) that is characterized by reduced theta power in the electroencephalogram (EEG) spectra. The non-REM sleep (NREMS) EEG of each of the IL1R1 transgenic mouse strains also is characterized by enhanced power in the delta frequency band. In response to 6h of sleep deprivation, sleep of both IL1R1 transgenic mouse strains is more consolidated than that of WT animals. Additionally, the NREMS EEG of NSE-IL1R1 mice contains less delta power after sleep deprivation, suggesting astroglial IL1R1 activity may modulate sleep homeostasis. Intracerebroventricular injection of IL-1 fails to alter sleep or brain temperature of NSE-IL1R1 or GFAP-IL1R1 mice. These data suggest that selective IL1R1 expression on neurons or on astrocytes is not sufficient for centrally-administered IL-1 to induce sleep or fever. Lack of sleep and febrile responses to IL-1 in these IL1R1 transgenic mouse strains may be due to their inability to produce IL-6 in brain. Overall, these studies demonstrate, through the use of novel transgenic mice, that IL1R1 on neurons and astrocytes differentially mediates aspects of sleep under physiological conditions and in response to central IL-1 administration.

Sepsis-induced alterations in sleep of rats.

Sepsis is a systemic immune response to infection that may result in multiple organ failure and death. Polymicrobial infections remain a serious clinical problem, and in the hospital, sepsis is the number-one noncardiac killer. Although the central nervous system may be one of the first systems affected, relatively little effort has been made to determine the impact of sepsis on the brain. In this study, we used the cecal ligation and puncture (CLP) model to determine the extent to which sepsis alters sleep, the EEG, and brain temperature (Tbr) of rats. Sepsis increases the amount of time rats spend in non-rapid eye movement sleep (NREMS) during the dark period, but not during the light period. Rapid eye movements sleep (REMS) of septic rats is suppressed for about 24 h following CLP surgery, after which REMS increases during dark periods for at least three nights. The EEG is dramatically altered shortly after sepsis induction, as evidenced by reductions in slow-frequency components. Furthermore, sleep is fragmented, indicating that the quality of sleep is diminished. Effects on sleep, the EEG, and Tbr persist for at least 84 h after sepsis induction, the duration of our recording period. Immunohistochemical assays focused on brain stem mechanisms responsible for alterations in REMS, as little information is available concerning infection-induced suppression of this sleep stage. Our immunohistochemical data suggest that REMS suppression after sepsis onset may be mediated, in part, by the brain stem GABAergic system. This study demonstrates for the first time that sleep and EEG patterns are altered during CLP-induced sepsis. These data suggest that the EEG may serve as a biomarker for sepsis onset. These data also contribute to our knowledge of potential mechanisms, whereby infections alter sleep and other central nervous system functions.

Preliminary outcomes of the Lake Erie College of Osteopathic Medicine's 3-year Primary Care Scholar Pathway in osteopathic predoctoral education.

CONTEXT: In 2007, the Lake Erie College of Osteopathic Medicine initiated its Primary Care Scholar Pathway (PCSP), a 3-year osteopathic predoctoral education curriculum. OBJECTIVE: To assess preliminary outcomes of the PCSP curriculum. METHODS: Scores for the Comprehensive Osteopathic Medical Licensing Examination (COMLEX-USA) Levels 1 and 2-Cognitive Evaluation (CE) and pass rates for Level 2-Performance Evaluation (PE) were obtained for individuals who graduated from the PCSP program in 2010, 2011, and 2012. Scores for Levels 1 and 2-CE were compared with national mean scores. Acceptance rates for residency programs were also recorded. RESULTS: Nineteen PCSP graduates were included in the study: 3 graduated in 2010, 6 graduated in 2011, and 10 graduated in 2012. Scores for PCSP students were not significantly different than national average scores for COMLEX-USA Levels 1 and 2-CE (P>.05). All 19 PCSP graduates passed the COMLEX-USA Level 2-PE on the first attempt, and all graduates were accepted into primary care residency programs. CONCLUSION: The COMLEX-USA scores of PCSP graduates were similar to national mean scores, suggesting that it is possible for osteopathic medical students to attain the same level of education as students of 4-year programs in less time. A 3-year osteopathic predoctoral education curriculum would allow students to complete their education at a reduced cost. This potential reduction in debt burden could encourage more students to pursue a primary care career and thus could help address the shortage of primary care physicians in the United States.

Sepsis-induced morbidity in mice: effects on body temperature, body weight, cage activity, social behavior and cytokines in brain.

Infection negatively impacts mental health, as evidenced by the lethargy, malaise, and cognitive deficits experienced during illness. These changes in central nervous system processes, collectively termed sickness behavior, have been shown in animal models to be mediated primarily by the actions of cytokines in brain. Most studies of sickness behavior to date have used bolus injection of bacterial lipopolysaccharide (LPS) or selective administration of the proinflammatory cytokines interleukin-1ß (IL-1ß) or IL-6 as the immune challenge. Such models, although useful for determining mechanisms responsible for acute changes in physiology and behavior, do not adequately represent the more complex effects on central nervous system (CNS) processes of a true infection with replicating pathogens. In the present study, we used the cecal ligation and puncture (CLP) model to quantify sepsis-induced alterations in several facets of physiology and behavior of mice. We determined the impact of sepsis on cage activity, body temperature, food and water consumption and body weights of mice. Because cytokines are critical mediators of changes in behavior and temperature regulation during immune challenge, we also quantified sepsis-induced alterations in cytokine mRNA and protein in brain during the acute period of sepsis onset. We now report that cage activity and temperature regulation in mice that survive are altered for up to 23 days after sepsis induction. Food and water consumption are transiently reduced, and body weight is lost during sepsis. Furthermore, sepsis decreases social interactions for 24-48 h. Finally, mRNA and protein for IL-1ß, IL-6, and tumor necrosis factor-α (TNFα) are upregulated in the hypothalamus, hippocampus, and brain stem during sepsis onset, from 6h to 72 h post sepsis induction. Collectively, these data indicate that sepsis not only acutely alters physiology, behavior and cytokine profiles in brain, but that some brain functions are impaired for long periods in animals that survive.

Fatal hemorrhage in mice lacking gamma-glutamyl carboxylase.

The carboxylation of glutamic acid residues to gamma-carboxyglutamic acid (Gla) by the vitamin K-dependent gamma-glutamyl carboxylase (gamma-carboxylase) is an essential posttranslational modification required for the biological activity of a number of proteins, including proteins involved in blood coagulation and its regulation. Heterozygous mice carrying a null mutation at the gamma-carboxylase (Ggcx) gene exhibit normal development and survival with no evidence of hemorrhage and normal functional activity of the vitamin K-dependent clotting factors IX, X, and prothrombin. Analysis of a Ggcx(+/-) intercross revealed a partial developmental block with only 50% of expected Ggcx(-/-) offspring surviving to term, with the latter animals dying uniformly at birth of massive intra-abdominal hemorrhage. This phenotype closely resembles the partial midembryonic loss and postnatal hemorrhage previously reported for both prothrombin- and factor V (F5)-deficient mice. These data exclude the existence of a redundant carboxylase pathway and suggest that functionally critical substrates for gamma-carboxylation, at least in the developing embryo and neonate, are primarily restricted to components of the blood coagulation cascade.

Transcription factor SIX5 is mutated in patients with branchio-oto-renal syndrome.

Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by the association of branchial arch defects, hearing loss, and renal anomalies. Mutations in EYA1 are known to cause BOR. More recently, mutations in SIX1, which interacts with EYA1, were identified as an additional cause of BOR. A second member of the SIX family of proteins, unc-39 (SIX5), has also been reported to directly interact with eya-1 in Caenorhabditis elegans. We hypothesized that this interaction would be conserved in humans and that interactors of EYA1 represent good candidate genes for BOR. We therefore screened a cohort of 95 patients with BOR for mutations in SIX5. Four different heterozygous missense mutations were identified in five individuals. Functional analyses of these mutations demonstrated that two mutations affect EYA1-SIX5 binding and the ability of SIX5 or the EYA1-SIX5 complex to activate gene transcription. We thereby identified heterozygous mutations in SIX5 as a novel cause of BOR.

Regulation of c-Ret in the developing kidney is responsive to Pax2 gene dosage.

During kidney development, Pax2 and Pax8 are expressed very early in the mammalian nephric duct and both precede the expression of receptor tyrosine kinase, c-Ret. However, in Pax2-/- mutant mice, expression of c-Ret is lost after embryonic day 10.5. As the Ret/Gdnf pathway is necessary for renal development and there is a temporal and spatial relationship of Pax2 and c-Ret expression in the developing genito-urinary system, we postulate that Pax2 is necessary for c-Ret expression in the developing kidney. In vitro, Pax2 protein is capable of physically interacting with a c-RET promoter, and both Pax2 and Pax8 can activate the expression of a reporter gene driven by the c-RET promoter. Compound heterozygous null mice (Pax2+/-: Ret+/-) display an increased incidence of unilateral and bilateral renal agenesis, and smaller kidneys with fewer nephrons. Furthermore, the expression of Gdnf is reduced 2-3-fold, whereas c-Ret expression is reduced 9-47-fold in Pax2 heterozygous embryonic kidneys as detected by real-time quantitative RT (QRT)-PCR. The data demonstrate that Pax2 plays an integral role in the initiation and maintenance of the Ret/Gdnf pathway by not only activating the ligand of the pathway, but by also enhancing the expression of the pathway receptor Ret. The effects of reduced Pax2 gene dosage are thus amplified resulting in a haploinsufficient phenotype.

SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes.

Urinary tract malformations constitute the most frequent cause of chronic renal failure in the first two decades of life. Branchio-otic (BO) syndrome is an autosomal dominant developmental disorder characterized by hearing loss. In branchio-oto-renal (BOR) syndrome, malformations of the kidney or urinary tract are associated. Haploinsufficiency for the human gene EYA1, a homologue of the Drosophila gene eyes absent (eya), causes BOR and BO syndromes. We recently mapped a locus for BOR/BO syndrome (BOS3) to human chromosome 14q23.1. Within the 33-megabase critical genetic interval, we located the SIX1, SIX4, and SIX6 genes, which act within a genetic network of EYA and PAX genes to regulate organogenesis. These genes, therefore, represented excellent candidate genes for BOS3. By direct sequencing of exons, we identified three different SIX1 mutations in four BOR/BO kindreds, thus identifying SIX1 as a gene causing BOR and BO syndromes. To elucidate how these mutations cause disease, we analyzed the functional role of these SIX1 mutations with respect to protein-protein and protein-DNA interactions. We demonstrate that all three mutations are crucial for Eya1-Six1 interaction, and the two mutations within the homeodomain region are essential for specific Six1-DNA binding. Identification of SIX1 mutations as causing BOR/BO offers insights into the molecular basis of otic and renal developmental diseases in humans.