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Biomedical subjects

J F Miller

Publications and source records attributed to J F Miller.

At least 19 recordsLinked to original sources
PubMed2006

Species- and strain-specific control of a complex, flexible regulon by Bordetella BvgAS.

The Bordetella master virulence regulatory system, BvgAS, controls a spectrum of gene expression states, including the virulent Bvg(+) phase, the avirulent Bvg(-) phase, and at least one Bvg-intermediate (Bvg(i)) phase. We set out to define the species- and strain-specific features of this regulon based on global gene expression profiling. Rather than functioning as a switch, Bvg controls a remarkable continuum of gene expression states, with hundreds of genes maximally expressed in intermediate phases between the Bvg(+) and Bvg(-) poles. Comparative analysis of Bvg regulation in B. pertussis and B. bronchiseptica revealed a relatively conserved Bvg(+) phase transcriptional program and identified previously uncharacterized candidate virulence factors. In contrast, control of Bvg(-)- and Bvg(i)-phase genes diverged substantially between species; regulation of metabolic, transporter, and motility loci indicated an increased capacity in B. bronchiseptica, compared to B. pertussis, for ex vivo adaptation. Strain comparisons also demonstrated variation in gene expression patterns within species. Among the genes with the greatest variability in patterns of expression, predicted promoter sequences were nearly identical. Our data suggest that the complement of transcriptional regulators is largely responsible for transcriptional diversity. In support of this hypothesis, many putative transcriptional regulators that were Bvg regulated in B. bronchiseptica were deleted, inactivated, or unregulated by BvgAS in B. pertussis. We propose the concept of a "flexible regulon." This flexible regulon may prove to be important for pathogen evolution and the diversification of host range specificity.

Bacterial Proteins↗
PubMed2001

Construction of mini-Tn5cyaA' and its utilization for the identification of genes encoding surface-exposed and secreted proteins in Bordetella bronchiseptica.

A mini-Tn5 transposon derivative, mini-Tn5cyaA', has been constructed. It contains a promoter-less and ribosome binding site-deficient reporter gene, encoding the catalytic domain of Bordetella pertussis adenylate cyclase toxin (CyaA'). We used this system to mutagenize B. bronchiseptica and we developed a screen for identification of mutants containing cyaA' translational fusions. This system was used to identify B. bronchiseptica genes that encode surface-exposed and secreted proteins.

Adenylate Cyclase Toxin↗
PubMed2001

Mechanisms of Bordetella pathogenesis.

Bordetella are Gram negative bacteria that cause respiratory tract infections in humans and animals. While at least five different species of Bordetella are known to exist, this review focuses on B. pertussis, B. bronchiseptica and B. parapertussis subspecies. In their virulent phase, all of these bacteria produce a nearly identical set of virulence factors which include adhesins such as filamentous hemagglutinin (FHA), fimbriae and pertactin, as well as toxins such as a bifunctional adenylate cyclase/hemolysin, dermonecrotic toxin, tracheal cytotoxin, a B. pertussis specific pertussis toxin and B. bronchiseptica specific type III secreted proteins. Expression of nearly all of these virulence factors is positively regulated by the products of the bvgAS locus. BvgA and BvgS comprise a two-component signal transduction system that mediates transition between at least three identifiable phases---a virulent (Bvg+) phase, an avirulent (Bvg-) phase and an intermediate (Bvg(i)) phase---in response to specific environmental signals. Bordetella colonize the ciliated respiratory mucosa, a surface designed to eliminate foreign particles, thereby making the adherence and persistence mechanisms of these bacteria crucial. The development of relevant animal models for B. bronchiseptica has enabled us to study Bordetella pathogenesis in the context of natural host-pathogen interactions. In addition, evolutionary studies across the various Bordetella species and detailed analysis of differential regulation of Bvg-activated/repressed genes has greatly enhanced our understanding of the mechanisms of Bordetella pathogenesis.

Animals↗
PubMed2001

Immune self-tolerance mechanisms.

Tolerance of both T and B cells to self-antigens can be achieved through a great variety of different routes, at the level of the primary lymphoid organs (thymus and bone marrow) and throughout the secondary lymphoid tissues. Whether self-reactive lymphocytes ignore their target autoantigen, or are tolerized by the various mechanisms discussed, depends on the circumstances.

Animals↗
PubMed2001

Ligand density determines the efficiency of negative selection in the thymus.

To study the influence of antigen density on the efficiency of negative selection in the thymus, MHC class I (H-2K(b), K(b)) transgenic mice were generated, which expressed a K(b) transgene under the control of its natural promoter at 33% (K(b-lo)) or 150% (K(b-hi)) the surface density of Kb in C57BL/6 (B6, H-2(b)) mice. These mice were crossed to anti-K(b) T-cell receptor (Des-TCR) transgenic mice. In Des-TCRxK(b-hi) double transgenic mice, Des-TCR bearing T cells were completely eliminated during thymocyte maturation. In contrast, in Des-TCRxK(b-lo) double transgenic mice, two populations of Des-TCR T cells were evident, which either expressed the Des-TCR at intermediate density in the absence of CD8 (Des-TCR(int)CD8(-)) or expressed both the Des-TCR and CD8 at low density (Des-TCRloCD8lo). In the thymus of both types of double transgenic mice, no Des-TCR(+)CD4(+)CD8(+) thymocytes were detected, suggesting that deletion of Des-TCR cells occurred before the CD4(+)CD8(+) stage. Because only very few Des-TCR(+) thymocytes were found in Des-TCRxK(b-hi) transgenic mice, deletion of these T cells apparently occurred upon expression of the Des-TCR. By contrast, Des-TCRxK(b-lo) transgenic mice showed distinct populations of Des-TCR(int)CD4-8- and Des-TCR(lo)CD8(lo) thymocytes, suggesting that expression of the CD8 coreceptor was required to allow negative selection to proceed. Functional analyses showed that sublethally irradiated Des-TCRxK(b-lo) double transgenic mice were protected from lethal graft-versus-host disease by injected Des-TCR lymph node cells.

Animals↗
PubMed2001

Mice lacking the orphan G protein-coupled receptor G2A develop a late-onset autoimmune syndrome.

Mice with a targeted disruption of the gene encoding a lymphoid-expressed orphan G protein-coupled receptor, G2A, demonstrate a normal pattern of T and B lineage differentiation through young adulthood. As G2A-deficient animals age, they develop secondary lymphoid organ enlargement associated with abnormal expansion of both T and B lymphocytes. Older G2A-deficient mice (>1 year) develop a slowly progressive wasting syndrome, characterized by lymphocytic infiltration into various tissues, glomerular immune complex deposition, and anti-nuclear autoantibodies. G2A-deficient T cells are hyperresponsive to TCR stimulation, exhibiting enhanced proliferation and a lower threshold for activation. Our findings demonstrate that G2A plays a critical role in controlling peripheral lymphocyte homeostasis and that its ablation results in the development of a novel, late-onset autoimmune syndrome.

Animals↗
PubMed2001

Identification and characterization of BipA, a Bordetella Bvg-intermediate phase protein.

The Bordetella BvgAS sensory transduction system has traditionally been viewed as controlling a transition between two distinct phenotypic phases: the Bvg(+) or virulent phase and the Bvg(-) or avirulent phase. Recently, we identified a phenotypic phase of Bordetella bronchiseptica that displays reduced virulence in a rat model of respiratory infection concomitant with increased ability to survive nutrient deprivation. Characterization of this phase, designated Bvg-intermediate (Bvg(i)), indicated the presence of antigens that are maximally, if not exclusively, expressed in this phase and therefore suggested the existence of a previously unidentified class of Bvg-regulated genes. We now report the identification and characterization of a Bvg(i) phase protein, BipA (Bvg-intermediate phase protein A), and its structural gene, bipA. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that bipA is expressed maximally under Bvgi phase conditions and thus represents the first identified Bvgi phase gene. bipA encodes a 1578-amino-acid protein that shares amino acid sequence similarity at its N-terminus with the proposed outer membrane localization domains of intimin (Int) of enteropathogenic and enterohaemorrhagic Escherichia coli and invasin (Inv) of Yersinia spp. Although not apparent at the amino acid level, BipA is also similar to Int and Inv in that the proposed membrane-spanning domain is followed by several 90-amino-acid repeats and a distinct C-terminal domain. Localization studies using an antibody directed against the C-terminus of BipA indicated that its C-terminus is exposed on the bacterial cell surface. Western blot analysis with this same antibody indicated that BipA homologues are expressed in Bvg(i) phase Bordetella pertussis and Bordetella parapertussis. Comparison of a Delta bipA strain with wild-type B. bronchiseptica indicated that BipA is not required for Bvg(i) phase-specific aggregative adherence to rat lung epithelial cells in vitro or for persistent colonization of the rabbit respiratory tract in vivo. However, our data are consistent with the hypothesis that BipA, and the Bvg(i) phase in general, play an important role in the Bordetella infectious cycle, perhaps by contributing to aerosol transmission.

Adhesins, Bacterial↗
PubMed2001

Diversity in the Bordetella virulence regulon: transcriptional control of a Bvg-intermediate phase gene.

The BvgAS signal transduction system controls the expression of at least three distinct phenotypic phases that lie along a continuum of gene expression states. The Bvg+ phase is characterized by the expression of adhesins and toxins, whereas the Bvg- phase is characterized by motility in Bordetella bronchiseptica and the expression of vrg loci in Bordetella pertussis. The Bvg-intermediate (Bvgi) phase is characterized by the absence of Bvg-repressed phenotypes, the expression of some, but not all, Bvg-activated virulence factors and the presence of a recently discovered set of antigens and phenotypes that are unique to this phase. We report here the transcriptional regulation of bipA, the first-identified Bvgi phase gene. We have mapped the bipA promoter and identified numerous BvgA binding sites in the transcriptional control region. Based on these data, we present a model in which phase-dependent expression of bipA results from the spatial distribution and relative affinities of multiple BvgA binding sites relative to the start site of transcription.

Adenylate Cyclase Toxin↗
PubMed2001

Effect of localized cytokine dysregulation: accelerated rejection of IL-2-expressing skin grafts.

Transgenic mice were created in which a sheep keratin promoter directed the expression of IL-2 into the dermis. These KIL-2 transgenic mice were used to investigate the effects of localized IL-2 dysregulation on immune responses. Peripheral tolerance to skin antigens was not broken by in situ IL-2 expression because syngeneic KIL-2 skin grafts were not rejected. However, MHC Class I-disparate skin grafts from KIL-2 donors were rejected faster (median survival time (MST) 12 days) than grafts of non-transgenic littermate skin (MST 18 days). In contrast, the kinetics of KIL-2 H-Y-disparate skin graft rejection (MST 14 days) did not differ significantly from controls (MST 16 days), suggesting that upregulation of IL-2 at the effector site could affect CD4+ T cell- independent, but not CD4+ T cell-dependent, responses. No effect on rejection kinetics was observed when wild type allogeneic skin was grafted onto transgenic mice that expressed bcl2 constitutively in their lymphocytes (MST of 14 days, both sets), indicating that this was not simply due to increased longevity of T cells within the IL-2 expressing graft. We therefore suggest that aberrant expression of IL-2 can accelerate helper-independent CD8+ T cell responses by increasing proliferation and/or differentiation of cytolytic T cells at the effector site.

Animals↗
PubMed2001

Kidney protection against autoreactive CD8(+) T cells distinct from immunoprivilege and sequestration.

BACKGROUND: The kidney tubulointerstitium has been reported to be protected from T-cell--mediated damage by sequestration from the T-cell compartment. We examined the ability of autoreactive T cells to infiltrate the kidney in a transgenic mouse model. METHODS: RIP-mOVA transgenic mice express the model autoantigen, membrane-bound ovalbumin (mOVA), in kidney proximal tubular cells and pancreatic beta cells. OVA-specific CD8(+) T cells (OT-I cells) were transferred into these recipient mice and their immune response against pancreas and kidney tissue was compared. RESULTS: When OVA-specific CD8(+) T cells (OT-I cells) were injected into RIP-mOVA mice, they were activated in the renal and pancreatic lymph nodes by cross-presentation. These in vivo-activated OT-I cells caused the destruction of pancreatic islets leading to autoimmune diabetes, but did not infiltrate the kidney. Neither CD95--CD95 ligand interactions, which have been proposed to induce apoptosis in T cells infiltrating immunologically privileged sites, nor CD30 signaling was responsible for the lack of kidney infiltration. When OT-I cells were activated in vitro prior to injection, they could infiltrate the kidney and caused acute renal failure when injected in high numbers. CONCLUSIONS: A mechanism distinct from previously described organ-specific protective mechanisms such as sequestration of antigen or CD95-mediated immunoprivilege contributes to the protection of the kidney tubulointerstitium from infiltration by autoreactive CD8(+) T cell.

Animals↗
PubMed2001

Spinal interneurons that receive input from muscle afferents are differentially modulated by dorsolateral descending systems.

The possibility that descending systems have differential actions on the spinal interneurons that receive input from muscle afferents was investigated. Prolonged, physiological inputs were generated by stretch of the triceps surae muscles. The resulting firing patterns of 25 lumbosacral interneurons were recorded before and during a reversible cold block of the dorsolateral white matter at the thoracic level in nonparalyzed, decerebrate preparations. The strength of group I muscle afferent input was assessed from the response to sinusoidal tendon vibration, which activated muscle spindle Ia afferents directly and tendon organ Ib afferents via the resulting reflex force. The stretch-evoked responses of interneurons with strong responses to vibration were markedly suppressed by dorsal cold block, whereas the stretch-evoked responses of interneurons with weak vibration input were enhanced. The cells most strongly activated by vibration received their primary input from Ia afferents and all of these cells were inhibited by the cold block. These results suggest that a disruption of the descending system, such as occurs in spinal cord injury, will lead to a suppression of the interneuronal pathways with group Ia input while enhancing excitability within interneuronal pathways transmitting actions from higher threshold afferents. One possible consequence of this suppression would be a decreased activity among the Ia inhibitory interneurons that mediate reciprocal inhibition, resulting in abnormal reciprocal relations between antagonists and promoting anomalous muscle cocontraction.

Animals↗
PubMed2000

Manipulating the host to study bacterial virulence.

The ability to manipulate animal hosts as well as bacterial pathogens greatly expands the utility of in vivo models of infection. For example, the construction of mice that harbor human tissues or express specific transgenes can provide ligand-receptor interactions that are essential for pathogenesis. Interactions between virulence factors and specific host defenses can sometimes be resolved by challenging selectively immuno deficient mice with bacteria containing virulence gene mutations. Transgenic animals expressing inducible reporters can be used to conveniently identify cells in which specific response pathways have been activated during infection. These and other approaches promise to improve the quality of information obtainable from in vivo assessments of pathogenesis.

Animals↗
PubMed2000

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system.

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti-Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-kappaB into large complexes in the cytoplasm. NF-kappaB aggregation was dependent on type III secretion and aggregated NF-kappaB did not respond to TNFalpha activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-kappaB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.

Animals↗
PubMed2000

Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection.

The present report provides the first extensive characterization of the OT-I TCR transgenic line, which produces MHC class I-restricted, ovalbumin-specific, CD8+ T cells (OT-I cells). These cells are shown to be positively selected in vivo in H-2b C57BL/6 mice and in bm5 mice, which express the Kbm5 mutant molecule. In contrast, OT-I cells were not selected by mutant Kb molecules in bm1, bm3, bm8, bm10, bm11 or bm23 mice. Interestingly, however, when positive selection was examined in vitro in foetal thymic organ culture (FTOC), bm1 and bm8 were still poorly selective, but the bm3 haplotype now selected as efficiently as B6. The ability to select in vitro correlated with the capacity to present the ovalbumin (OVA) peptide to OT-I cells, as measured by induction of an OVA-specific proliferative response. These results suggest that a lower affinity TCR:MHC interaction may be necessary for positive selection in FTOC compared with selection in situ.

Animals↗
PubMed2000

Betahistine effects on cochlear blood flow: from the laboratory to the clinic.

The development of laser Doppler flowmetry techniques has contributed greatly to the study of cochlear blood flow (CBF). In animal models, intravenous betahistine dihydrochloride clearly increased CBF in a dose-dependent manner. This effect was greater in the cochlear vasculature than in the systemic vascular bed. The effects of betahistine were blocked by the alpha 2-antagonist idazoxan, thus suggesting an interaction between histaminergic and presynaptic adrenergic receptors. This was further supported by studies investigating the effects of electrical stimulation on CBF. Local (round window membrane) application of betahistine did not affect CBF, but had a non-specific effect on cochlear electrophysiology. This indicates that the receptors for betahistine vascular effects in the inner ear are most likely located in the modiolar artery. More recently, laser Doppler flowmetry techniques have been applied to human subjects. It has been shown that intratympanic application of adrenaline affects CBF and that this blood flow is under vigorous sympathetic control. Electrical stimulation has also been used to obtain measures of dynamic responsiveness in human subjects. This results in an increase in CBF, which is dependent on the intensity of the stimulation. Preliminary evidence indicates that this procedure can provide a standardized measure of the dynamic properties of CBF and may provide a means to differentially identify patients with compromised vasculature.

Adrenergic Agonists↗