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Biomedical subjects

J F Miller

Publications and source records attributed to J F Miller.

At least 91 records · Page 5Linked to original sources
PubMed1996

Restoration of extensor excitability in the acute spinal cat by the 5-HT2 agonist DOI.

1. The decerebrate cat preparation with an intact spinal cord is characterized by a high degree of excitability in extensor motoneuron pools, which is eliminated by acute spinalization. Subtype-specific agonists for serotonin (5-HT) were investigated in terms of their effectiveness in restoring the extensor excitability following spinalization. 2. Our hypothesis was that 5-HT2 receptors have the primary role in enhancement of extensor reflex excitability, whereas 5-HT1A and 5-HT1B/D receptors are relatively unimportant. Reflex excitability was assessed from the tonic levels of force and electromyographic (EMG) output from the ankle extensors medial gastrocnemius (MG) and soleus (SOL), and from the reflex forces in both these muscles generated by ramp-and-hold stretches of MG. 3. Before spinal transection, MG and SOL usually exhibited a small amount of tonic background EMG activity and force output. Ramp-and-hold stretch of MG generated a large-amplitude reflex response. Spinal transection at the level of T10 virtually abolished tonic background activity in both extensors and greatly attenuated the MG stretch reflex. Ventral topical application of the selective 5-HT2A/2C agonist (+-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane hydrochloride (DOI) restored the amplitude of the MG stretch reflex in a dose-dependent fashion. However, a considerable portion of the DOI-mediated restoration of MG stretch reflex force was due to elevation of tonic background force levels above previous intact cord levels. 4. The DOI-induced increase in extensor tonic background excitability and facilitation of MG stretch reflex were reversed by ventral topical administration of the selective 5-HT2 antagonist ketanserin. No increase in extensor excitability was observed in spinalized preparations after administration of either the 5-HT1A agonist (+-)-8-hydroxy-dipropylaminotetralin hydrobromide or the 5-HT1B/1D agonist 7-trifluoromethyl-4-(4 methyl-1-piperazinyl)-pyrrolo[1,2- a]quinoxaline maleate. These data strongly suggest that the DOI-induced facilitation of extensor stretch reflex and tonic activity in spinalized preparations is mediated through an action on spinal 5-HT2 receptors. 5. One important difference between the actions of DOI in spinalized versus intact states was that the DOI-induced tonic and reflex forces in the spinalized state were subject to irregular oscillations. In contrast, DOI did not noticeably affect the smoothness of reflex force generation in the intact state. This discrepancy was probably due to the effects of clasp knife inhibition from muscular free nerve endings, which have potent reflex actions in the spinalized but not intact states. Thus DOI elevated excitability levels but did not alter the effects of spinalization on stretch reflex patterns.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1996

Genetic regulation of airway colonization by Bordetella species.

Bordetella species are respiratory pathogens that infect humans and other animals. A majority of the virulence factors expressed by these bacteria are regulated by a master control locus, BvgAS, a member of the two-component family of signal transduction systems. BvgAS senses environmental signals and mediates signal transduction by a phosphorylation cascade that leads to a biphasic transition between the Bvg+ and Bvg- phases. From natural host studies using Bordetella bronchiseptica, we have found that expression of Bvg+ phase factors, which include adhesins and toxins, is required for successful colonization of the mammalian respiratory tract. Suppression of the Bvg- phase motility phenotype is necessary for a successful interaction with the host. Although the Bvg- phase does not appear to be required in vivo, it does confer the ability to survive under conditions of severe nutrient deprivation. We hypothesize that the Bvg+ phase is necessary and sufficient for respiratory tract colonization and the Bvg- phase is adapted for survival in environments encountered during transmission between hosts.

Animals↗
PubMed1995

Deletion of high-avidity T cells by thymic epithelium.

Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helper-independent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kb-specific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of those CD8+ T cells with the highest density of the clonotypic TCR. These cells presumably represent the T cells with the highest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.

Animals↗
PubMed1995

Autoimmunity caused by ignorant CD8+ T cells is transient and depends on avidity.

RIP-Kb mice, which express H-2Kb (Kb) molecules on their pancreatic beta cells, were used to examine the requirements for induction of autoimmune diabetes caused by CD8+ T cells. Previous studies showed that when these mice were crossed to mice expressing a Kb-specific TCR transgene, those CD8+ cells expressing the highest density of the transgenic TCR (presumably the highest avidity cells) were deleted intrathymically due to aberrant expression of Kb at this site. The remaining low avidity cells ignored Kb-bearing beta cells, even after priming, but were able to cause autoimmune diabetes when supplied with Il-2. To examine the properties of high avidity autoreactive CD8+ T cells, the thymic compartment of RIP-Kb mice was replaced with normal tissue to enable the maturation of CD8+ cells expressing the highest density of the transgenic TCR. These high avidity cells generally ignored Kb-expressing beta cells, but became autoaggressive after priming. Importantly, analysis of islet infiltration by CD8+ T cells revealed the presence of infiltrating cells in all mice examined within 3 wk of priming, but such infiltration was not usually apparent at later time points. In some cases, multiple primings were necessary for full development of autoimmunity. This implied that beta cells could act as transient targets for CD8+ T cell attack but could not sustain the stimulation of primed CD8+ cells. These studies indicate that the duration of priming stimulus and the avidity of the autoreactive CD8+ cells profoundly influence the severity of autoimmune disease.

Animals↗
PubMed1995

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity.

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.

Amino Acid Sequence↗
PubMed1995

Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction.

Signal transduction molecules within the two-component family represent a conserved adaptation for the control of genes involved in pathogenesis. The Bordetella virulence control locus, bvgAS, activates and represses gene expression in response to environmental signals. While infection requires virulence gene activation, the role of gene repression during infection is not understood. By altering regulatory genes and reversing regulatory connections, we found evidence that the BvgAS-repressed genes responsible for motility are neither required nor expressed during colonization of the host. Expression of this Bvg- phase-specific phenotype in the Bvg+ growth phase resulted in a defect in tracheal colonization. Therefore, BvgAS promotes virulence both by activating genes required for colonization and by repressing genes that inhibit the development of infection.

Animals↗
PubMed1995

Expression of two T cell receptor alpha chains on the surface of normal murine T cells.

We have previously reported that a subset of T cells in T cell receptor (TCR)-transgenic mice may express two different alpha chains on their surface. The expression of two functional alpha chains has also been demonstrated for human peripheral blood T cells. In this report, we show that a proportion of normal murine lymph node T cells express two functional alpha chains on their surface. The extrapolated frequency of these cells present in the normal repertoire ranges from 7-21%, with an average of 15%. Our analysis of a small number of antigen-specific T cell clones suggests that the frequency of antigen-responsive cells expressing two surface alpha chains is relatively low. This raises the possibility that dual alpha chain T cells may have a selective disadvantage in responding to specific antigen.

Amino Acid Sequence↗
PubMed1995

Peripheral deletion of autoreactive CD8+ T cells in transgenic mice expressing H-2Kb in the liver.

The response of T cells specific for liver antigens was examined in transgenic mice expressing the allogeneic major histocompatibility complex class I molecule H-2Kb (Kb) under the control of the sheep metallothionein promoter (Met-Kb mice). To follow the fate of Kb-specific T cells, and to prevent any aberrant thymic expression of the Kb transgene, the mice were thymectomized, lethally irradiated, protected with bone marrow cells from transgenic mice expressing in their T cells a Kb-specific T cell receptor identifiable by a clonotypic antibody, and given syngeneic non-transgenic thymus grafts. Although Kb-specific CD8+ T cells were produced in the thymus grafts of these manipulated Met-Kb mice, only small numbers of such cells could be detected in the spleen and lymph nodes. The livers, however, showed signs of damage and were heavily infiltrated by actively dividing CD8+ T cells. We provide strong evidence that the hepatocytes, not generally regarded as antigen-presenting cells, activated the Kb-specific CD8+ T cells and that these disappeared after a vigorous autoimmune response that resulted in deletion.

Animals↗
PubMed1995

Effect of reversible dorsal cold block on the persistence of inhibition generated by spinal reflexes.

The effects of bilateral focal cooling of dorsolateral thoracic spinal cord on segmental reflex pathways to the triceps surae muscles were assessed in decerebrate cats from the reflex forces produced by single shocks or trains of electrical stimuli applied to the ipsilateral caudal cutaneous sural and the contralateral tibial nerves. The validity of the dorsal cold block technique as a substitute for acute surgical dorsal hemisection was established by showing that focal cooling reliably reproduced the stretch-induced "clasp knife" inhibition of triceps surae reflexive force seen following dorsal hemisection. Under control (warm) conditions, the inhibitory components of electrically evoked ipsilateral sural and contralateral tibial reflexes faded rapidly during sustained trains, with a resultant production of large-amplitude reflex force as measured from either the entire triceps surae or from the medial gastrocnemius muscle alone. Dorsal cold block greatly reduced the amplitude of reflexive force evoked by sustained electrical stimulation of either nerve. Indeed, the cold block completely reversed the sign of train-evoked reflexes to a net inhibition of reflex force output in one-half of the sural and one-half of the contralateral tibial stimulation experiments. Peak transient forces evoked by single shocks to the sural or contralateral tibial nerves were also sometimes reduced, but this result was more variable than for prolonged nerve stimulation. The persistence of activity in segmental inhibitory pathways during dorsal cold block, as indicated by instances of reflex sign reversal, suggests that descending bulbospinal pathways traversing the dorsolateral funiculi may be responsible for "fading" of segmental inhibitory reflex components in decerebrate cats with intact spinal cords during sustained afferent input. The possibility that the enhanced magnitude and duration of segmental inhibition during cold block will increase the likelihood of disruption of the size principle for motoneuron recruitment is also discussed.

Animals↗
PubMed1995

Validity of parent report measures of vocabulary development for children with Down syndrome.

This study investigated the validity of a parent report measure of vocabulary development, the MacArthur Communicative Development Inventory: Words and Sentences (CDI), in children with and without developmental disabilities. Concurrent validity was examined by comparing results from the CDI and laboratory measures of vocabulary in 44 children with Down syndrome and 46 typically developing children with mental ages from 12 to 27 months. Significant correlations between .70 and .82 were obtained. Predictive validity was examined by measuring the vocabulary of 20 children with Down syndrome and 23 typically developing children first at approximately 20 months mental age and later at a mental age of approximately 28 months. Significant correlations were obtained between the CDI at Time A and all but one of the vocabulary measures at Time B (r = .46 to .66). These results establish the validity of parent measures of vocabulary development for children with Down syndrome and confirm their validity for typically developing children.

Child↗
PubMed1995

bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage.

Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.

Bacterial Proteins↗
PubMed1995

BvgAS is sufficient for activation of the Bordetella pertussis ptx locus in Escherichia coli.

BvgA and BvgS, which regulate virulence gene expression in Bordetella pertussis, are members of the two-component signal transduction family. The effects of growth conditions on the ability of BvgAS to activate transcription of fhaB (encoding filamentous hemagglutinin) and ptxA (encoding the S1 subunit of pertussis toxin) were assessed in Escherichia coli by using chromosomal fhaB-lacZYA and ptxA-lacZYA fusions. Although it had previously been reported that a ptxA-lacZYA transcriptional fusion was not activated by bvgAS in E. coli (J. F. Miller, C. R. Roy, and S. Falkow, J. Bacteriol. 171:6345-6348, 1989), we now present evidence that ptxA is activated by bvgAS in E. coli in a manner that is highly dependent on the growth conditions. Higher levels of beta-galactosidase were produced by ptxA-lacZYA in the presence of bvgAS during growth in Stainer-Scholte medium or M9 minimal salts medium with glucose than in Luria-Bertani medium. In contrast, the level of fhaB-lacZYA expression was high during growth in all media. Addition of modulating stimuli which inhibit BvgAS function eliminated expression of ptxA-lacZYA. Levels of beta-galactosidase expressed from the ptx-lacZYA fusion correlated with growth rate and with the final optical density at 600 nm, suggesting that the lower growth rate in M9-glucose and Stainer-Scholte media was responsible for greater accumulation of beta-galactosidase than was seen in Luria-Bertani medium. Overproduction of BvgA was not sufficient for activation of ptxA expression but was sufficient for fhaB expression. However, overproduction of a constitutive BvgA allele (bvgA-Cl) or overproduction of BvgA in the presence of BvgS was able to activate ptxA. Our results demonstrate Bvg-dependent activation of a ptxA-lacZYA fusion in E. coli and indicate that bvg is the only Bordetella locus required for ptxA activation in this heterologous system.

Adhesins, Bacterial↗
PubMed1995

5-HT1B/1D agonist CGS-12066B attenuates clasp knife reflex in the cat.

1. The effect of intrathecal injection of the selective serotonin (5-HT)1B/1D receptor agonist CGS-12066B maleate (825 nmol) was assessed on stretch-evoked clasp knife inhibition of hindlimb ankle extensor muscle reflex force in precollicular decerebrate cats in which neural transmission in dorsolateral spinal pathways was blocked bilaterally by focal cooling. 2. During cold block, ramp and hold stretches of the medial gastrocnemius muscle (MG) evoked only a brief reflex excitation that was followed by powerful, long-lasting inhibition (the clasp knife reflex). Both the amplitudes of peak force evoked by the ramp and sustained force output during the last 500 ms of the hold phase of the stretch were depressed by > 50%. 3. Reflex force output during the hold portion of stretch was significantly improved on postdrug cold block trials, although peak force remained depressed. CGS-12066B did not significantly alter stretch-evoked force output in decerebrate cats when spinal cord neural transmission was unimpaired. 4. These data suggest that selective 5-HT1B/1D agonists may be of therapeutic usefulness in the treatment of reflex disorders arising from partial spinal cord injury.

Animals↗
PubMed1995

Focal expression of interleukin-2 does not break unresponsiveness to "self" (viral) antigen expressed in beta cells but enhances development of autoimmune disease (diabetes) after initiation of an anti-self immune response.

The participation of IL-2 in insulin-dependent (type 1) diabetes (IDDM) was analyzed in transgenic (tg) mice expressing the nucleoprotein (NP) of lymphocytic choriomeningitis virus and IL-2 under control of the rat insulin promoter focally in beta cells of the islets of Langerhans. Insertion and expression of the viral (self) gene or of the IL-2 gene alone did not lead to IDDM. Infiltration primarily of CD4 and B lymphocytes and increased expression of MHC class I and II molecules occurred in islets where IL-2 was expressed. By contrast, neither cellular infiltrates nor expression of MHC class I or II glycoproteins above base levels was noted in tgs expressing the viral protein alone. Double tg mice expressing both the viral protein and IL-2 in their islets displayed a modest increase in incidence of spontaneous diabetes compared with that of single transgenic mice expressing IL-2 alone. Breaking of immunological unresponsiveness or sensitization to self antigens did not occur. Neither cytotoxic T lymphocytes (CTL) nor antibodies directed against the viral tg (NP) were generated. However, after challenge with lymphocytic choriomeningitis virus, double tg mice developed anti-self (viral) CTL and IDDM (incidence > 95%) within 2 mo. The generation of virus ("self")-specific MHC-restricted CTL was dependent on CD4+ help. In contrast, viral inoculum to single tg mice expressing either the viral protein or IL-2 failed to enhance the incidence of IDDM over 30% for viral protein or 10% for IL-2 after an 8-mo observation period. Hence, in this autoimmune model in situ expression of IL-2 did not break unresponsiveness but markedly enhanced ongoing disease.

Analysis of Variance↗
PubMed1995

Autoantigen-induced deletion of peripheral self-reactive T cells.

There are now numerous reports documenting the deletion of mature peripheral specific T cells following massive antigenic stimulation. Such a phenomenon can be regarded as a homeostatic mechanism to prevent unrestricted growth of antigen-activated clones and to safeguard against autoimmunity. Some of the reports examining this issue are summarized in this review and the role of the liver in tolerance and autoimmunity is discussed based on recent work performed with transgenic mice.

Animals↗
PubMed1995

Individual differences in vocabulary acquisition in children with Down syndrome.

Children with Down syndrome offer an opportunity for investigators interested in cognition and language problems to study asynchronous development of vocabulary, and syntax in language production relative to comprehension and cognitive status. Our work to date has documented consistent differences in the rate of vocabulary learning in children with DS relative to their mental age matched peers. These deficits increase with advancing age indicating that the learning rate of the DS group is significantly slower than that of the TD group. While the rate of vocabulary acquisition is significantly slower for the children with DS, not all of the children we studied exhibited similar rates of learning. Thirty-five percent of our children had rates of vocabulary growth consistent with mental age expectations. These data suggest that neither the syndrome alone nor the cognitive deficit associated with Down syndrome can explain the differences observed in vocabulary growth. Our next series of studies will evaluate a variety of causal constructs to explain the significant asynchronies in language learning compared with other cognitive abilities.

Down Syndrome↗
PubMed1994

Autophosphorylation and phosphotransfer in the Bordetella pertussis BvgAS signal transduction cascade.

Expression of adhesins, toxins, and other virulence factors of Bordetella pertussis is under control of the BvgA and BvgS proteins, members of a bacterial two-component signal transduction family. BvgA bears sequence similarity to regulator components, whereas BvgS shows similarity to both sensor and regulator components. BvgA and the cytoplasmic portion of BvgS ('BvgS) were overexpressed and purified. 'BvgS autophosphorylated with the gamma-phosphate from [gamma-32P]ATP and phosphorylated BvgA. Kinetic analysis indicated that BvgA receives its phosphate from 'BvgS. Mutations in the transmitter, receiver, and C-terminal domains of BvgS were tested for activation of a BvgAS-dependent fhaB::lacZ reporter fusion in vivo and for autophosphorylation and phosphotransfer to BvgA in vitro. All mutations abolished activation of the fhaB::lacZ fusion. A point mutation in the transmitter (H729Q) prevented autophosphorylation of 'BvgS. In contrast to other characterized sensor proteins, autophosphorylation also required sequences in the 'BvgS receiver and C-terminal domains. A 'BvgS receiver point mutation (D1023N) had the novel phenotype of being able to autophosphorylate but unable to transfer the phosphate to BvgA. Autophosphorylation activity of the D1023N mutant protein was kinetically and chemically indistinguishable from wild-type 'BvgS despite an uncoupling of phosphotransfer from autophosphorylation. 'BvgS was shown to contain primarily amidyl phosphate and BvgA an acyl phosphate linkage. We present a model for a phosphorelay controlling virulence gene expression in B. pertussis.

Bacterial Proteins↗