Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1.

Familial hemiplegic migraine type 1 (FHM1) is an autosomal dominant subtype of migraine with aura that is associated with hemiparesis. As with other types of migraine, it affects women more frequently than men. FHM1 is caused by mutations in the CACNA1A gene, which encodes the alpha1A subunit of Cav2.1 channels; the R192Q mutation in CACNA1A causes a mild form of FHM1, whereas the S218L mutation causes a severe, often lethal phenotype. Spreading depression (SD), a slowly propagating neuronal and glial cell depolarization that leads to depression of neuronal activity, is the most likely cause of migraine aura. Here, we have shown that transgenic mice expressing R192Q or S218L FHM1 mutations have increased SD frequency and propagation speed; enhanced corticostriatal propagation; and, similar to the human FHM1 phenotype, more severe and prolonged post-SD neurological deficits. The susceptibility to SD and neurological deficits is affected by allele dosage and is higher in S218L than R192Q mutants. Further, female S218L and R192Q mutant mice were more susceptible to SD and neurological deficits than males. This sex difference was abrogated by ovariectomy and senescence and was partially restored by estrogen replacement, implicating ovarian hormones in the observed sex differences in humans with FHM1. These findings demonstrate that genetic and hormonal factors modulate susceptibility to SD and neurological deficits in FHM1 mutant mice, providing a potential mechanism for the phenotypic diversity of human migraine and aura.

Activity of anidulafungin in a murine model of Candida krusei infection: evaluation of mortality and disease burden by quantitative tissue cultures and measurement of serum (1,3)-beta-D-glucan levels.

Experience with anidulafungin against Candida krusei is limited. Immunosuppressed mice were injected with 1.3 x 10(7) to 1.5 x 10(7) CFU of C. krusei. Animals were treated with saline, 40 mg/kg fluconazole, 1 mg/kg amphotericin B, or 10 and 20 mg/kg anidulafungin for 5 days. Anidulafungin improved survival and significantly reduced the number of CFU/g in kidneys and serum beta-glucan levels.

Quantitative diffusion tensor imaging detects dopaminergic neuronal degeneration in a murine model of Parkinson's disease.

Early diagnosis of Parkinson's disease (PD) is required to improve therapeutic responses. Indeed, a clinical diagnosis of resting tremor, rigidity, movement and postural deficiencies usually reflect >50% loss of the nigrostriatal system in disease. In a step to address this, quantitative diffusion tensor magnetic resonance imaging (DTI) was used to assess nigrostriatal degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication model of dopaminergic nigral degeneration. We now demonstrate increased average diffusion (p<0.005) and decreased fractional anisotropy (p<0.03) in the substantia nigra (SN) of 5- to 7-day MPTP-treated animals when compared to saline controls. Transverse diffusivity demonstrated the most significant differences (p < or = 0.002) and correlated with the numbers of SN dopaminergic neurons (r=-0.75, p=0.012). No differences were found in the striatum, corpus callosum, cerebral cortex, or ventricles. These results demonstrate that DTI may be used as a surrogate biomarker of nigral dopaminergic neuronal degeneration.

The effects of ultraviolet B radiation on immune responses to {\it Borrelia burgdorferi}, the causative agent of Lyme disease

Ultraviolet B (UVB) radiation, in addition to being carcinogenic, is also immunosuppressive. Immunologically, UVB induces suppression locally, at the site of irradiation, or systemically, by inducing the production of a variety of immunosuppressive cytokines. Systemic effects include suppression of delayed-type hypersensitivity (DTH) responses to a variety of antigens (e.g. haptens, proteins, bacterial antigens, or alloantigens). One of the principal mediators of UV-induced immune suppression is the T helper-2 (Th2) cytokine interleukin-10 (IL-10); this suggests that UV irradiation induces suppression by shifting the immune response from a Th1 (cellular) to a Th2 (humoral) response. These "opposing" T helper responses are usually mutually exclusive, and polarized Th1 or Th2 responses may lead to either protection from infection or increased susceptibility to disease, depending on the infectious agent and the route of infection.^ This study examines the effects of UVB irradiation on cellular and humoral responses to Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD) in both immunization and infectious disease models; in addition, it examines the role of T cells in protection from and pathology of Bb infection. Particular emphasis is placed on the Bb-specific antibody responses following irradiation since UVB effects on humoral immunity are not fully understood. Mice were irradiated with a single dose of UV and then immunized (in complete Freund's adjuvant) or infected with Bb (intradermally at the base of the tail) in order to examine both DTH and antibody responses in both systems. UVB suppressed the Th1-associated antibodies IgG2a and IgG2b in both systems, as well as the DTH response to Bb in a dose dependent manner. Injection of anti-IL-10 antibody into UV-irradiated mice within 24 h after UV exposure restored the DTH response, as well as the Th1 antibody (IgG2a and IgG2b) response. In addition, injecting recombinant IL-10 mimicked some of the effects of UV radiation.^ Bb-specific Th1 T cell lines (BAT2.1-2.3) were generated to examine the role of T cells in Lyme borreliosis. All lines were CD4$\sp+,$ $\alpha\beta\sp+$ and proliferated specifically in response to Bb. The BAT2 cell lines not only conferred a DTH response to naive C3H recipients, but reduced the number of organisms recovered from the blood and tissues of mice infected with Bb. Furthermore, BAT2 cell lines protected mice from Bb-induced periarthritis. ^

Detecting genetic and nutritional lung cancer risk factors related to folate metabolism using Bayesian generalized linear models

Complex diseases, such as cancer, are caused by various genetic and environmental factors, and their interactions. Joint analysis of these factors and their interactions would increase the power to detect risk factors but is statistically. Bayesian generalized linear models using student-t prior distributions on coefficients, is a novel method to simultaneously analyze genetic factors, environmental factors, and interactions. I performed simulation studies using three different disease models and demonstrated that the variable selection performance of Bayesian generalized linear models is comparable to that of Bayesian stochastic search variable selection, an improved method for variable selection when compared to standard methods. I further evaluated the variable selection performance of Bayesian generalized linear models using different numbers of candidate covariates and different sample sizes, and provided a guideline for required sample size to achieve a high power of variable selection using Bayesian generalize linear models, considering different scales of number of candidate covariates. ^ Polymorphisms in folate metabolism genes and nutritional factors have been previously associated with lung cancer risk. In this study, I simultaneously analyzed 115 tag SNPs in folate metabolism genes, 14 nutritional factors, and all possible genetic-nutritional interactions from 1239 lung cancer cases and 1692 controls using Bayesian generalized linear models stratified by never, former, and current smoking status. SNPs in MTRR were significantly associated with lung cancer risk across never, former, and current smokers. In never smokers, three SNPs in TYMS and three gene-nutrient interactions, including an interaction between SHMT1 and vitamin B12, an interaction between MTRR and total fat intake, and an interaction between MTR and alcohol use, were also identified as associated with lung cancer risk. These lung cancer risk factors are worthy of further investigation.^

Mutual Information for Testing Gene-Environment Interaction

Despite current enthusiasm for investigation of gene-gene interactions and gene-environment interactions, the essential issue of how to define and detect gene-environment interactions remains unresolved. In this report, we define gene-environment interactions as a stochastic dependence in the context of the effects of the genetic and environmental risk factors on the cause of phenotypic variation among individuals. We use mutual information that is widely used in communication and complex system analysis to measure gene-environment interactions. We investigate how gene-environment interactions generate the large difference in the information measure of gene-environment interactions between the general population and a diseased population, which motives us to develop mutual information-based statistics for testing gene-environment interactions. We validated the null distribution and calculated the type 1 error rates for the mutual information-based statistics to test gene-environment interactions using extensive simulation studies. We found that the new test statistics were more powerful than the traditional logistic regression under several disease models. Finally, in order to further evaluate the performance of our new method, we applied the mutual information-based statistics to three real examples. Our results showed that P-values for the mutual information-based statistics were much smaller than that obtained by other approaches including logistic regression models.

Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.

We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC(fm), is transcribed as an operon, that its putative major pilus subunit, EbpC(fm) (also called pilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC(fm) operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC(fm) expression and eliminated EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), was not affected. Importantly, the ebpABC(fm) deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC(fm) in trans, which also restored cell surface expression of EbpC(fm) and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC(fm) locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.

Importance of the collagen adhesin ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis.

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFDeltaace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFDeltaace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.

Importance of the ebp (endocarditis- and biofilm-associated pilus) locus in the pathogenesis of Enterococcus faecalis ascending urinary tract infection.

BACKGROUND: We recently demonstrated that the ubiquitous Enterococcus faecalis ebp (endocarditis- and biofilm-associated pilus) operon is important for biofilm formation and experimental endocarditis. Here, we assess its role in murine urinary tract infection (UTI) by use of wild-type E. faecalis OG1RF and its nonpiliated, ebpA allelic replacement mutant (TX5475). METHODS: OG1RF and TX5475 were administered transurethrally either at an ~1 : 1 ratio (competition assay) or individually (monoinfection). Kidney pairs and urinary bladders were cultured 48 h after infection. These strains were also tested in a peritonitis model. RESULTS: No differences were observed in the peritonitis model. In mixed UTIs, OG1RF significantly outnumbered TX5475 in kidneys (P=.0033) and bladders (P< or =.0001). More OG1RF colony-forming units were also recovered from the kidneys of monoinfected mice at the 4 inocula tested (P=.015 to P=.049), and 50% infective doses of OG1RF for kidneys and bladder (9.1x10(1) and 3.5x10(3) cfu, respectively) were 2-3 log(10) lower than those of TX5475. Increased tropism for the kidney relative to the bladder was observed for both OG1RF and TX5475. CONCLUSION: The ebp locus, part of the core genome of E. faecalis, contributes to infection in an ascending UTI model and is the first such enterococcal locus shown to be important in this site.

Brain fiber tract plasticity in experimental spinal cord injury: diffusion tensor imaging.

Diffusion tensor imaging (DTI) and immunohistochemistry were performed in spinal cord injured rats to understand the basis for activation of multiple regions in the brain observed in functional magnetic resonance imaging (fMRI) studies. The measured fractional anisotropy (FA), a scalar measure of diffusion anisotropy, along the region encompassing corticospinal tracts (CST) indicates significant differences between control and injured groups in the 3 to 4 mm area posterior to bregma that correspond to internal capsule and cerebral peduncle. Additionally, DTI-based tractography in injured animals showed increased number of fibers that extend towards the cortex terminating in the regions that were activated in fMRI. Both the internal capsule and cerebral peduncle demonstrated an increase in GFAP-immunoreactivity compared to control animals. GAP-43 expression also indicates plasticity in the internal capsule. These studies suggest that the previously observed multiple regions of activation in spinal cord injury are, at least in part, due to the formation of new fibers.

Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Endocarditis and biofilm-associated pili of Enterococcus faecalis.

Increasing multidrug resistance in Enterococcus faecalis, a nosocomial opportunist and common cause of bacterial endocarditis, emphasizes the need for alternative therapeutic approaches such as immunotherapy or immunoprophylaxis. In an earlier study, we demonstrated the presence of antibodies in E. faecalis endocarditis patient sera to recombinant forms of 9 E. faecalis cell wall-anchored proteins; of these, we have now characterized an in vivo-expressed locus of 3 genes and an associated sortase gene (encoding sortase C; SrtC). Here, using mutation analyses and complementation, we demonstrated that both the ebp (encoding endocarditis and biofilm-associated pili) operon and srtC are important for biofilm production of E. faecalis strain OG1RF. In addition, immunogold electron microscopy using antisera against EbpA-EbpC proteins as well as patient serum demonstrated that E. faecalis produces pleomorphic surface pili. Assembly of pili and their cell wall attachment appeared to occur via a mechanism of cross-linking of the Ebp proteins by the designated SrtC. Importantly, a nonpiliated, allelic replacement mutant was significantly attenuated in an endocarditis model. These biologically important surface pili, which are antigenic in humans during endocarditis and encoded by a ubiquitous E. faecalis operon, may be a useful immunotarget for studies aimed at prevention and/or treatment of this pathogen.

Acute and chronic methylphenidate dose-response assessment on three adolescent male rat strains.

Methylphenidate (MPD), commonly known as Ritalin, is the most frequently prescribed drug to treat children and adults with attention deficit hyperactivity disorder (ADHD). Adolescence is a period of development involving numerous neuroplasticities throughout the central nervous system (CNS). Exposure to a psychostimulant such as MPD during this crucial period of neurodevelopment may cause transient or permanent changes in the CNS. Genetic variability may also influence these differences. Thus, the objective of the present study was to determine whether acute and chronic administration of MPD (0.6, 2.5, or 10.0mg/kg, i.p.) elicit effects among adolescent WKY, SHR, and SD rats and to compare whether there were strain differences. An automated, computerized, open-field activity monitoring system was used to study the dose-response characteristics of acute and repeated MPD administration throughout the 11-day experimental protocol. Results showed that all three adolescent rat groups exhibited dose-response characteristics following acute and chronic MPD administration, as well as strain differences. These strain differences depended on the MPD dose and locomotor index. Chronic treatment of MPD in these animals did not elicit behavioral sensitization, a phenomenon described in adult rats that is characterized by the progressive augmentation of the locomotor response to repeated administration of the drug. These results suggest that the animal's age at time of drug treatment and strain/genetic variability play a crucial role in the acute and chronic effect of MPD and in the development of behavioral sensitization.

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.